Abstract
The fibroproliferative reaction to acute lung injury may limit restoration of normal lung function and increase mortality in patients with acute lung injury. A biologic marker of collagen synthesis in the lung may be useful for studying the pathogenesis of acute lung injury and for identifying patients with acute lung injury who are at high risk for death and might benefit from new therapeutic modalities. Using an immunoassay, type III procollagen NH2 terminal peptide was measured in the pulmonary edema fluid of 44 patients with either acute lung injury or hydrostatic pulmonary edema (control group) within the first 24 h after endotracheal intubation for acute respiratory failure. Patients with acute lung injury (n = 33) or hydrostatic edema (n = 11) had the same degree of lung dysfunction as measured by the severity of oxygenation defect, the level of positive end-expiratory pressure, the decrease in static lung compliance, and the extent of infiltrates on the chest radiograph. However, the median procollagen III level was 5-fold higher in the pulmonary edema fluid of patients with acute lung injury than in the patients with hydrostatic pulmonary edema (p = 0.0001). Of the 33 patients with acute lung injury, 21 patients died and 12 lived. Nonsurvivors had significantly higher procollagen III levels than did survivors (p = 0.05). The positive and negative predictive values for nonsurvival for a procollagen III level ⩾ 1.75 U/ml were 74 and 83%, respectively. The relative risk of dying in the presence of a procollagen III value ⩾ 1.75 U/ml was 4.5 (95% CI, 0.7 to 27). Collagen synthesis in the lung, as reflected by elevated levels of procollagen III in pulmonary edema fluid, begins within the first 24 h of acute lung injury concurrent with the acute phase of increased endothelial and epithelial permeability to protein. This evidence suggests that fibrosing alveolitis begins much earlier in the course of clinical acute lung injury than has previously been appreciated. In addition, the presence of an elevated level of procollagen III is an early predictor of poor outcome. Thus, elevation of procollagen III in pulmonary edema fluid may have both pathogenetic and prognostic significance in patients with acute lung injury.
Fibrosing alveolitis is a well recognized consequence of acute lung injury and may contribute to protracted respiratory failure and death in many patients with the acute respiratory distress syndrome (ARDS). On the basis of pathologic studies, several investigators have concluded that fibrosing alveolitis develops subacutely around Days 5 to 7 after acute lung injury (1-3). Increased levels of collagen, the major extracellular matrix component of lung, are present in the lungs of patients with acute lung injury (4, 5). Immunohistologic evaluation of lung tissue from patients with ARDS demonstrates an abundance of type I and type III collagen (6).
Collagen is synthesized primarily by lung fibroblasts as a procollagen precursor molecule. Collagen accumulation in ARDS results, at least in part, from increased procollagen synthesis, a mechanism that has been studied in several animal models of acute lung injury (4, 7). The N-terminal peptide of type III procollagen, cleaved from the precursor procollagen molecule by specific proteinases in the extracellular space (8), has been used as a biologic marker of collagen synthesis. In experimentally induced wound healing, procollagen III in serum reflects fibrogenesis (9-11). Elevated levels have been detected in the serum from patients with hepatic cirrhosis (12), wound-healing (11), and trauma (13). In patients with lung disease, elevated procollagen III levels are found in serum and brochoalveolar lavage of patients with sarcoidosis (14), interstitial pulmonary fibrosis (15), Pneumocystis carinii pneumonia (16), and ARDS (17). Recently, elevated procollagen III levels were detected in the bronchoalveolar lavage fluid obtained at 3, 7, and 14 d after the onset of ARDS in a majority of patients (18). Elevated levels were associated with an increased risk for death independent of other known risk factors and physiologic measures of severity. Although procollagen III was not detected in bronchoalveolar lavage from normal volunteers, no comparison group of patients with acute respiratory failure from cardiogenic or hydrostatic pulmonary edema was studied. Furthermore, that study did not examine patients at the onset of acute lung injury.
Therefore, the primary objective of this study was to measure procollagen peptide III levels in the pulmonary edema fluid of patients at the onset of acute lung injury to test the hypotheses that the initial phase of fibrosing alveolitis begins very early in the course of acute lung injury. Because there is a major need for early reliable biologic markers of acute lung injury to guide clinical trials of patients with acute lung injury, the second objective was to determine if elevations of procollagen peptide III in pulmonary edema fluid would have prognostic value for predicting mortality in patients with acute lung injury.
This study was approved by the Committee on Human Research at the University of California, San Francisco. Patients were enrolled from the four adult intensive care units at the University of California at San Francisco, Moffitt-Long Hospital. The patients were identified prospectively by one of the critical care fellows or attendings within the first 24 h of endotracheal intubation. In the vast majority of patients, edema fluid and blood samples were collected within 1 h of intubation or within 1 h of the development of radiographic evidence of pulmonary edema. The criteria for enrollment was endotracheal intubation and the ability to obtain pulmonary edema fluid. Patients were excluded if edema fluid was unobtainable within the first 24 h. There also were patients with pulmonary edema who did not have fluid collected, usually because the patient was admitted during hours when a fellow or attending was not available to collect the edema fluid.
At the time this study was initiated, we randomly selected pulmonary edema fluid samples from 44 patients collected over a 10-yr period, from 1986 to 1995. We did not attempt to select samples according to patient demographics, clinical history, total protein values or other information.
To obtain edema fluid, a sterile 14-gauge suction catheter was inserted through the endotracheal tube and wedged into the distal airways to obtain an undiluted sample of pulmonary edema fluid, as previously described (19-22). We do not know exactly where the catheter wedges in the lung and, more importantly, it is not yet known what the reproducibility of measurements may be if repeated sampling is done in the same patient over brief time intervals. Sequential samples over time can be done with this method (21), although they were not done in this study. Simultaneous blood samples also were collected. After collection, the sample was centrifuged at 3,000 g and the total protein was measured by the biuret method in the edema fluid and in the plasma (22). Aliquots of the fluid were frozen at −70° C and coded without reference to the patient's clinical history.
Patients were classified as having acute lung injury (increased permeability pulmonary edema) if their initial edema fluid to plasma total protein ratio was ⩾ 0.75. Hydrostatic edema was defined as a ratio ⩽ 0.65, as we have previously reported (19-22). The classification of hydrostatic edema was confirmed by evidence of left ventricular dysfunction on a two-dimensional echocardiogram, or an elevated pulmonary arterial occlusion (wedge) pressure (> 18 mm Hg). Furthermore, we determined that all patients with increased permeability edema met the North American-European Consensus Conference definition for acute lung injury: acute onset of illness, bilateral infiltrates on the chest radiograph, a PaO2 /Fi O2 ratio < 300, a pulmonary arterial wedge pressure less than 18 mm Hg, or no clinical evidence of left atrial hypertension (23).
Survival was defined as survival to hospital discharge. The primary cause of death was evaluated using criteria similar to that used in a recent large clinical trial in patients with acute lung injury (24). Respiratory failure was the primary cause of death when it was not possible to ventilate or oxygenate the patient adequately on maximal ventilator support. Sepsis was the cause of death when severe systemic hypotension occurred in the presence of maximal vasopressor support. Multiple organ dysfunction was the primary cause of death when there was simultaneous failure of three or more organ systems.
The medical chart was reviewed to determine the primary risk factors for ARDS. The criteria for the diagnosis of sepsis syndrome were a temperature ⩾ 38° C or ⩽ 35° C, a systolic blood pressure < 90 mm Hg, and a clinically identifiable source of infection. Pneumonia was defined as evidence of primary lung infection from bacterial, viral, or fungal infection diagnosed by Gram's stain and culture of tracheal aspirate or bronchoalveolar lavage specimens. Patients who simultaneously met criteria for both sepsis syndrome and pneumonia were classified as septic. Patients who developed acute lung injury in association with other clinical disorders such as multiple blood transfusions, aspiration, or drug overdose were classified as “other.” Major trauma victims are not cared for at UCSF Moffitt-Long Hospital.
The lung injury score, determined at the time of endotracheal intubation, was based on a four-point scoring system that includes evaluation of the chest radiograph, arterial oxygenation, respiratory system compliance, and the level of positive end-expiratory pressure (PEEP). This scoring system has been described in detail elsewhere (25) and used by us (26, 27) and other investigators (28).
The concentration of procollagen III in coded edema fluid specimens or serum was determined by radioimmunoassay (CIS-US, Inc., Bedford, MA) using 20 μl of edema fluid or serum. The radioimmunoassay for procollagen peptide III was linear over a range of 0.4 to 9.5 U/ml. Serum control samples provided by the manufacturer contained 1.6 to 1.7 U/ml. Samples in which the procollagen III concentration was greater than the standard detection range were diluted 1:4 in 0.9% NaCl. Samples in which procollagen III concentration was below the detection range were assigned a value of 0.4 U/ml for subsequent data analysis. In a prior study, normal volunteers were used for controls for measurements of procollagen III (18), but in this study controls consisted of those patients with hydrostatic pulmonary edema. We thought that these patients would represent better controls because they had severe respiratory failure from edema in the lung and required positive-pressure ventilation. The need for positive-pressure ventilation in the setting of severe hydrostatic pulmonary edema may predispose to some low level of inflammation that might result in release of procollagen by the lung.
Categorical variables were analyzed using a chi-square analysis (Fisher's exact test). Continuous variables were analyzed using an unpaired t test. Procollagen III levels in pulmonary edema fluid samples (hyrdrostatic versus acute lung injury) were analyzed using a nonparametric test, the Mann Whitney U test. Because procollagen levels in survivors and nonsurvivors had unequal variances, an unpaired t test was done after log transformation of the data.
Because a retrospective analysis of procollagen III levels in bronchoalveolar lavage had demonstrated that levels more than 1.75 U/ml were associated with nonsurvival in patients with ARDS (18), we prospectively tested this cutoff as a predictor of mortality. Standard formulas were used to calculate positive predictive value, sensitivity and specificity, relative risk, and confidence intervals.
Thirty-three patients were identified with increased permeability edema and 11 patients with hydrostatic edema based on the edema fluid to plasma total protein ratio and clinical data (Table 1). All 33 patients with increased permeability edema met the North American-European definition of acute lung injury; six of 33 patients met the criteria for acute lung injury (PaO2 /Fi O2 ⩽ 300), and 27 of 33 met the criteria for ARDS (PaO2 /Fi O2 ⩽ 200). The two groups were similar in age and sex. The physiologic indices of the severity of respiratory dysfunction, including the PaO2 /Fi O2 ratio and the lung injury score, were nearly identical in the patients with acute lung injury and the control patients with hydrostatic edema (Table 1). The major clinical disorders associated with the development of acute lung injury were sepsis and pneumonia (n = 22). Other clinical disorders such as gastric aspiration, drug overdose, and massive transfusions were present in 11 patients.
| Characteristic | Acute Lung Injury(n = 33) | Hydrostatic Edema(n = 11) | ||
|---|---|---|---|---|
| Age, yr* | 53 (21–93) | 66 (22–91) | ||
| Men/Women, n/n | 16/17 | 5/6 | ||
| PaO2 /Fi O2 † | 127 ± 66 | 132 ± 68 | ||
| Lung injury score (day 1)†,‡ | 2.76 ± 0.57 | 2.53 ± 0.49 | ||
| Edema fluid/plasma protein ratio† | 0.93 ± 0.29§ | 0.54 ± 0.12 |
The median procollagen III level was 5-fold higher in the acute lung injury group than in the hydrostatic edema group (median, 3.12 versus 0.6 U/ml, p = 0.001) (Figure 1).
Fig. 1. Pulmonary edema fluid procollagen III concentrations in patients with lung injury edema and hydrostatic edema. Median procollagen III level was higher in patients with lung injury edema (hatched column) than in patients with hydrostatic edema (open column) within 24 h of intubation (p = 0.0001). Horizontal lines indicate the median value. Colulmns represent the 25th to 75th percentile. Vertical lines represent the 10th to 90th percentile. Individual values below the 10th percentile and above the 90th percentile are shown as open circles.
[More] [Minimize]The median plasma procollagen III levels were 1.8 and 0.63 U/ml in the acute lung injury and hydrostatic groups, respectively (p = 0.0002). There was no correlation between the pulmonary edema fluid and the plasma procollagen III values (r = 0.12). In fact, the three patients with the highest edema fluid values (35.5, 48.2, and 150) had serum levels of only 1.6, 2.0, and 2.8 U/ml, respectively.
Within the group of 33 patients with acute lung injury, 21 died (64%), and 12 lived (36%). Nonsurvivors were significantly older and more likely to be male (Table 2). On Day 1, the two groups had comparable degrees of lung injury and impairment of oxygenation. The primary causes of death in the 21 nonsurvivors were respiratory failure (38%), sepsis (24%), multiple organ dysfunction syndrome (24%), cardiac arrest (5%), and other (9%). However, all the patients who died continued to have ongoing ventilator-dependent respiratory failure at the time of death.
| Clinical Data | Survivors(n = 12) | Nonsurvivors(n = 21) | p Value | |||
|---|---|---|---|---|---|---|
| Age, yr | 45 | 59 | 0.002 | |||
| Men/women | 2/10 | 13/8 | 0.008 | |||
| Primary diagnosis | ||||||
| Sepsis | 5 | 11 | NS | |||
| Pneumonia* | 2 | 7 | NS | |||
| Other | 5 | 3 | NS | |||
| PaO2 /Fi O2 † | 149 ± 53 | 114 ± 70 | 0.14 | |||
| Lung injury score†,‡ | 2.6 ± 0.6 | 2.8 ± 0.6 | 0.24 | |||
| Procollagen III, units/ml§ | 2.5 | 3.7 | 0.05 |
The median procollagen III level in nonsurvivors (4.0 U/ml) was significantly higher than in survivors (2.5 U/ml) (p = 0.05) (Figure 2). The range in the nonsurvivors was 0.95 to 150, whereas the range in the survivors was 0.2 to 19.5. To analyze the predictive value for nonsurvival of an elevated procollagen III level obtained at Day 1 of onset of ARDS, we used a cutoff of 1.75 U/ml. This level of procollagen III had been assigned retrospectively in a bronchoalveolar lavage study of patients with ARDS (18). Using this cutoff for nonsurvival, the sensitivity for predicting mortality was 95%, with a specificity of 42%. The positive predictive value for death of an elevated procollagen III level (> 1.75) was 74%, and the negative predictive value was 83% (Table 3). The relative risk for dying in the presence of procollagen III greater than 1.75 U/ml was 4.5 (95% CI, 0.7 to 27).
Fig. 2. Pulmonary edema fluid procollagen III concentrations in patients with acute lung injury who lived or died. Median procollagen III level was higher in patients with lung injury who died (hatched column) than in those who lived (open column) (p = 0.05). Horizontal lines indicate that median value. Columns represent the 25th to 75th percentile. Vertical lines represent the 10th to 90th percentile. Individual values below the 10th percentile and above the 90th percentile are shown as open circles.
[More] [Minimize]This study has provided important new evidence concerning the pathogenesis of clinical acute lung injury. Elevated levels of procollagen peptide III, a biologic marker of collagen synthesis, were detectable in the pulmonary edema fluid within the first 24 h after the clinical recognition of acute lung injury. These results indicate that fibrosing alveolitis may begin much earlier in the course of acute lung injury than was previously appreciated. It is likely that the release of procollagen III and the early production of collagen is a consequence of the severity of acute lung injury. In addition to the pathogenetic significance of this observation, elevated procollagen III levels identify those patients with an increased risk for death.
According to current concepts, the early phase of acute lung injury is characterized by an increase in pulmonary endothelial and epithelial permeability. Protein-rich edema fluid accumulates in the interstitial and air spaces of the lung, leading to arterial hypoxemia, decreased lung compliance, and increased work of breathing. This early phase is followed by a subacute fibroproliferative phase that may allow repair of injured lung or result in progressive obliteration of the interstitial and alveolar compartments of the lung (29). This study provides evidence that the fibroproliferative process may in fact be established when acute lung injury first becomes clinically apparent.
Our data clearly indicate that the elevated procollagen III levels were specific for lung injury and not merely correlated with severe pulmonary edema since patients with hydrostatic pulmonary edema of comparable severity according to physiologic indices had low or undetectable procollagen III levels in their edema fluid. Moreover, there was no relationship between the levels in pulmonary edema fluid and plasma from patients with ARDS, suggesting that the injured lung was the primary source of the procollagen III.
Although it is somewhat surprising that procollagen III levels were so markedly elevated very early in the course of ARDS, several lines of evidence support the concept that collagen synthesis may be stimulated rapidly after tissue injury. In a cutaneous wound-healing model, fibroblast activation and production of new collagen can be detected within 16 h (30). Several studies have shown that cytokines and growth factors that are known to activate fibroblasts are present in the edema fluid or bronchoalveolar lavage fluid of patients with ARDS within the first 24 h after onset of lung injury. For example, tumor necrosis factor (31), transforming growth factor-α (32), and interleukin-1 (33) have been detected early in the course of ARDS. Finally, collagen deposition detectable by histology or biochemical means is a process that requires at least several days to occur. It follows that development of pulmonary fibrosis within even a week of onset of ARDS would require an increase in lung collagen synthesis at the very early stages of acute lung injury.
The second major finding from this study is that the level of procollagen III in the edema fluid at the onset of ARDS has prognostic value. We found that nonsurvivors of acute lung injury had significantly higher procollagen III levels than did survivors. Previously, Clark and coworkers (18) reported that levels > 1.75 U/ml on Day 3 in bronchoalveolar lavage fluid were associated with a relative risk of 2.4. However, that cutoff was assigned retrospectively based on the median value. In this study, we examined this cutoff prospectively in a different population of patients with acute lung injury. In our patients, pulmonary edema fluid levels more than 1.75 U/ml were associated with a relative risk of 4.5. This cutoff value for procollagen III had a positive predictive value of 74% and a negative predictive value of 83% for nonsurvival.
Why is a biologic marker of collagen synthesis in the lung a predictor of mortality? The most obvious explanation is that increased procollagen III levels predict lung fibrosis and ongoing respiratory failure. The results of this study as well as several other reports indicate that mortality in patients with acute lung injury can be related to both nonpulmonary and pulmonary factors (18, 27, 34, 35). Some patients succumb to nosocomial infection or persistent multiple organ failure. However, the presence of persistent respiratory failure in acute lung injury is usually a central clinical problem (18, 24). In the present study, all patients had ongoing respiratory failure at the time of death. The association between lung fibrosis, acute lung injury, and death is also supported by a recent study that reported increased mortality rates in patients with biopsy evidence of pulmonary fibrosis (36). Elevated procollagen III in the edema fluid also may identify patients with a poor prognosis, not simply because all of these patients are destined to develop relentless respiratory failure from severe pulmonary fibrosis, but also because increased procollagen III synthesis is a marker of severe lung injury. In fact, in a prior study of procollagen III in bronchoalveolar lavage fluid, the quantity of protein in the air spaces was significantly higher in nonsurvivors than in survivors, suggesting that the extent of injury to the endothelial and epithelial barriers of the lung was greater (18).
The discovery that collagen synthesis begins within 24 h of lung injury and is not necessarily a subacute process significantly alters our current understanding regarding the pathogenesis of acute lung injury and suggests that future investigations should focus on the cellular and molecular mechanisms that modulate early activation of fibroblasts. Given the strong correlation between early collagen synthesis and death, future laboratory and clinical studies should focus on therapeutic modalities targeted at altering collagen synthesis early in the course of acute lung injury.
There are two limitations of this study. First, pulmonary edema fluid was not sampled from every patient who met acute lung criteria, and it is possible that the selected patients represent a subset with more severe lung injury. However, the edema fluids were obtained from a random sampling of patients over a 10-yr period, and the mortality of 64% in this group is not statistically different from the mortality of 58% from a recent series of 123 consecutive patients with acute lung injury from our institution (27). The second limitation is the lack of pathologic lung specimens in our patients to correlate with the procollagen III levels. Given the severity of illness in this patient population and the infrequent clinical indication for lung biopsy, early pathologic correlation in humans is difficult to obtain. In a recently published European study, 22 lung biopsy specimens were obtained from 25 consecutive patients with ARDS later in the clinical course (10 ± 3 d) (36). Fourteen patients (64%) had pathologic evidence of pulmonary fibrosis. Although the series was small, the mortality rate for the patients with pulmonary fibrosis was 57%, whereas all of the patients without fibrosis lived.
In conclusion, elevated levels of procollagen III, a biologic marker of collagen synthesis, are present in the pulmonary edema fluid from patients with acute lung injury. These elevated procollagen III levels are specific for acute lung injury since negligible concentrations were found in a control group of patients with severe respiratory failure from hydrostatic edema. Because the levels were markedly elevated on Day 1 of acute lung injury, it is likely that the process of fibrosing alveolitis begins very early in the course of acute lung injury. In addition to the pathogenetic significance of the elevated procollagen III levels, this biologic marker has potential practical value as an early predictor of nonsurvival in patients with acute lung injury.
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