Effect of Smoking on Pulmonary Vascular Permeability A Positron Emission Tomography Study1-3
JAMES D. KAPLAN, FRANK S. CALANDRINO, and DANIEL P. SCHUSTER4
Introduction
Several studies suggest that cigarette smoke can affect pulmonary vascular permeability. For example, cigarette smoke increases permeability to albumin flux in an endothelial cell monolayer (1). Epithelial permeability is also increased in chronic smokers as measured in vivo by clearance rates of [99mTc]-diethylenetriamine pentaacetic acid (DfPA) aerosol (2). These findings present a potential drawback to studying certain forms oflung disease because the specificity of an abnormal result is reduced if the subject smokes cigarettes. Nevertheless, epithelial permeability measurements by the 99mTc-DTPA clearance technique have been used to assess acute and chronic lung diseases, including pneumoconioses (3), idiopathic pulmonary fibrosis (4), inflammatory interstitial lung disease(5), and adult respiratory distress syndrome (ARDS) (6). The overlap in values between otherwise normal smokers and subjects with lung disease makes it difficult to interpret "abnormal" epithelial permeability in some patients. In previous studies with positron emission tomography (PET), we described a method to evaluate pulmonary vascular permeability by measuring the pulmonary transcapillary escape rate (PTCER) for radiolabeled transferrin. This noninvasive, repeatable method has been used to study different forms of acute lung injury, including experimental oleic acid lung injury (7), (ARDS) (8), and acute pneumonia (9). In experimental lung injury, abnormalities in PTCER correlate with histologic evidence of both endothelial and epithelial damage (10). However, if the PET technique could not adequately distinguish between abnormalities induced by smoking alone from those associated with ARDS, as is the case for the epithelial permeability measurement, its usefulness would clearly be reduced. Therefore, in the present study, we evaluated pulmonary vascular 712
SUMMARY Positron emission tomography (PET) can be used to evaluate pulmonary vascular endothelial permeability by measuring the pulmonary transcapiliary escape rate (PTCER) for radlolabeled transferrin. Because epithelial permeability, as evaluated by other techniques, Is significantly affected by cigarette smoking, we used PET to compare the effects of smoking on extravascular lung density (EVD)and PTCERin seven normal chronic cigarette smokers within 30 min of smoking a cigarette and seven normal nonsmokers. We found no difference in PTCERand EVD between the two groups. We conclude that the Interpretation of acute or chronic lung InJury studies with PET should not be affected by cigarette smoking In the subject population. AM REV RESPIR DIS 1992; 145:712-715
permeability in chronic smokers by measuring PTCER and compared these values with nonsmoking volunteer subjects with no evidence of lung disease. Methods Subjects We studied seven healthy nonsmoking adult volunteers and seven chronic cigarette smokers. Subjects were between 18 and 50 yr of age and were of either sex. Normal volunteers had no evidence by history or physical examination of active or prior cardiopulmonary disease. Smokers had smoked 10to 40 cigarettes daily for 3 to 15 pack-years and were asked to smoke a cigarette within 30 min of beginning the PET study. Despite their chronic cigarette use, smokers had no evidence of cardiopulmonary diseaseby medical history or physical examination.
Study Protocol The theory and methods of PET measurements have been reviewed recently (11, 12). PET scan data were acquired on the SUPERPETT I body scanner (13) and displayed as seven transaxial tomographic slices with a center-to-center separation of 15 mm and an intrinsic in-plant resolution of 18 mm. Subjects were studied in a supine position. A PET study involved collecting a background scan, a transmission scan, and a series of emission scans. The background and transmission scans wereperformed to correct emission scans for errors in the measurement of tissue activity introduced by tissue attenuation. Because activity attenuation is proportional to tissue density, the transmission scan also provided a density image resembling a
standard X-ray computed tomography image, which was used to define anatomic regions of interest. Serial emission scans were then collected for 60 min beginning 2 min after injection of up to 6 mCi of [68Gajcitrate, prepared according to previously published methods (7, 14, 15). Time-activity data were obtained for each region of interest in the lung and within the cardiac blood pool on each emission scan. These data were decay corrected to the time of tracer injection. The blood pool activity was analyzed by two sequential linear regressions to produce a biexponential expression for the fitted blood curve as previously described (7). The tissue and blood time-activitydata were analyzed with a two-compartment mathematical model. Using this model, the vascular-toextravascular rate constant k, was calculated. Since protein flux is affected by surface
(Received in original form February 11, 1991 and in revised form May 20" 1991) 1 From the Respiratory and Critical Care Medicine Division, Department of Internal Medicine, Washington University Medical School, St. Louis, Missouri. 2 Supported by Grant No. POI HL 13851 from the National Institutes of Health and Grant No. DE-FG02-87ER60512 from the Department of Energy. 3 Correspondence and requests for reprints should be addressed to Daniel Schuster, M.D., Respiratory and Critical Care Medicine Division, Box 8052, Washington University School of Medicine, St. Louis, MO 63110. 4 Established Investigator of the American Heart Association and Career Investigator of the American Lung Association.
713
PET STUDY OF SMOKING AND PULMONARY VASCULAR PERMEABILITY
Fig. 1. Single-slice images of total density, blood volume, extravascular density, and permeability from a representative study of a normal cigarette smoker. Lung regions and mean values for each lung are identified on each image.
area available for exchange, k, was normalized to regional blood volume, which is assumed to be proportional to this surface area. Regional plasma volume was determined by calculating the ratio of PET activity during the initial 2 min of data collection, when virtuallyall [68Ga]transferrin plasma label is still intravascular (7), to the corresponding integral of the fitted blood curve. Blood volume was determined from the plasma volume measurement, adjusting for hematocrit as measured by the capillary tube centrifuge method. PTCER wasthen calculated as k,/regional blood volume. The attenuation scan was scaled to the known density of blood so that the cardiac blood pool had a density of 1.05 g/ml (IS). This resulted in an image of regional lung density (rLD), including both the vascular and extravascular compartments. Regional extravascular density (EVD) was then calculated as rLD minus regional intravascular density.
PET measurements were expressed in the following units: EVD (g/IOO mllung), rLD (g/IOO ml lung), and PTCER (l0-4 mirr").
Image Analysis Lung regions were identified on trans axial slicesof the transmission scan for each subect, stored in computer memory, and then superimposed on images ofrLD, blood volume, EVD, and PTCER (figure I). Regions of interest included the entirety of the right and left lungs on all slices above the diaphragm, representing approximately 10.5 em of lung in a craniocaudal direction. Statistical Analysis The data are expressed as the mean ± SD. Analysis of variance (ANOVA) techniques were used to test for significant differences (p < 0.05) among the mean values. Within the ANOVA, t tests were conducted among previously chosen pairs of means to establish statistically significant differences. A
least-squares means analysis by the general linear model procedure of the StatisticalAnalysis System (SAS Institute, Cary, NC) for the IBM-PC was used for these statistical calculations.
Results
Mean age for nonsmoking and smoking subjects was not different (28.6 ± 8.3versus 30.4 ± 9.9, respectively, p = NS). Mean PTCER, rLO, and EVO were not different between smokers and nonsmokers (p = NS, figure 2). A small but statistically significant difference in rLO was present between the left and right sides within each group (nonsmokers p < 0.0025, smokers p < 0.0321,table 1). In addition, the nonsmokers' right lung rLO showed a small but statistically significant difference from the rLO of either lung in the smokers (p < 0.0025).
714
KAPLAN, CALANORINO, AND SCHUSTER
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Fig. 2. Mean ± SO permeability (PTCER), total lung density (rlO), and extravascular density (EVO) values for nonsmoking and smoking subjects. Open bars = nonsmokers; shaded bars = smokers.
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ing due to the position of the heart in the left hemithorax. There were also small density differences between the lowest value for nonsmokers (the right lung) and both the right and left lung rLD values for smokers. Since rLD is the sum of intravascular density plus EVD and EVD was not different between the two groups, this rLD increase is a reflection of an increased pulmonary capillary blood volume in the smokers 30 min after a cigarette. Because the bilaterally increased rLD in smokers was only statistically significant when compared with the least dense lung in nonsmokers, it is ofuncertain physiologic importance. Nevertheless, other studies have suggested that an increased density is plausible after cigarette smoking. For instance, nicotine is a well-known vasodilator and may be expected to cause increased regional blood volumes (18, 19). Studies of labeled erythrocytes and neutrophils demonstrate neutrophil retention in smokers' lungs (20, 21), and neutrophils and other cells are increased in lung capillaries for up to several days after exposure to cigarette smoke (21, 22). Our findings are also consistent with standard radiographic computed tomographic density measurements in smokers and nonsmokers (23, 24). On the other hand, our data are not explained by the peribronchial inflammation that can be present histologically in some chronic smokers (25). Changes due to inflammation, which are "ex-
The greatest magnitude of this difference was only 17"10 between this value for nonsmokers' right lung rLD and the densest average value (the smokers' left lung). No difference in PTCER or EVD was present between smokers and nonsmokers or between left and right sides within groups (table 1). Discussion
To determine the effect of cigarette smoking on the PTCER measurement as measured by PET, we evaluated endothelial permeability in otherwise normal smokers. The data are important because (1) permeability estimates by techniques that measure epithelial injury have been abnormal in otherwise normal smokers (16); (2) in vitro studies suggest that smoke causes abnormal permeability in endothelial tisue (1, 17); and (3) studies of subjects with acute or chronic lung disease frequently include smokers who may bias the results if smoking itself contributes to abnormal values of "permeability" (18). In this study, we found a small difference between the right and left lung total density in both smokers and nonsmokers. BecausePET data are expressed in terms of unit volume, regions of normal tissue can have higher density values simply as a result of atelectasis. Therefore, this biologically small elevation in rLD (up to 17%) on the left side in this study may reflect in part anatomic crowd-
TABLE 1 MEAN VALUES FOR NONSMOKERS ANO SMOKERS' EVO (g/100 ml)
rlO (g/100 ml)
Nonsmokers Smokers All subjects
Right
left
Mean
Right
left
35 ± 5t; 40 ± 5t 38 ± 4t
39 ± 6 42 ± 4 40 ± 5
37 ± 5 41 ± 4 39 ± 5
19 ± 5 19 ± 4 19 ± 4
17 ± 3 17 ± 4 17 ± 4
Mean
18 ± 4 18 ± 4 18 ± 4
PTCER (10-' min-f) Right
left
Mean
20 ± 12 22 ± 15 21 ± 9 15 ± 13 26 ± 16 21 ± 14 18 ± 13 24 ± 15 21 ± 11
• Mean ± SO values lor regional lung density (rLO), extravascular density (EVD), and permeability (PTCER) lor nonsmokers, smokers, and all subjects evaluated. t p < 0.05 right versus left. :; p < 0.05 lor right lung nonsmokers versus either lung smokers rLO values.
travascular," should be manifest as both increased EVD and rLD. Inflammation may be more relevant in patients with active chronic bronchitis (26), a group excluded by definition from this study. One other study used PET to measure density in normal smokers and nonsmokers (27). In a single tomographic slice at the midthoracic level (as opposed to an average from multiple contiguous slices in our study), EVD was 16% higher for smokers. Consistent with our data, total density was slightly greater in the left lung compared with the right lung, although this difference was not statistically significant. These differences between our data and this other study may be related to differences in the study populations, in the methods for defining regions of interest, or in the method for measuring blood volume. Although our own data in dogs suggest that 68Ga-Iabeled transferrin and CIsO-Iabeled red blood cells measure blood volume equally well (28), data in humans suggest that small differences between the two methods can occur (29). Overall, the magnitude of the density differences in either study, although sometimes statistically significant, were quite small. Because permeability measurements werenot performed in this other PET study, it is difficult to evaluate the importance of these differences in EVD. Some investigators have found the permeability to labeled albumin to be abnormal in endothelial cell monolayers exposed to cigarette smoke (1). It is difficult to compare our data with this in vitro effect. The in vivo dose of inhaled smoke may have been less than that used in vitro, or the levelof injury detected in vitro may be below the level of detection of our method. Moreover, direct comparisons suggest that there may be important differences between the permeability characteristics of endothelial cell monolayers and blood vessels in vivo (30) and between the permeability characteristics of endothelial and epithelial cell barriers as well (31). It is possible that an epithelial injury can bel detected by one method while the endothelial barrier remains intact. This would be consistent with the histologic effects of modest smoke exposure in guinea pigs, in which there is damage to epithelial but not endothelial structures (32, 33). Abnormalities are detectable in smokers for at least 7 days after smoking cessation by epithelial permeability techniques (34-37). Since epithelial permeability studies do not measure pulmonlary vascular endothelial injury per se,
PET STUDY OF SMOKING AND PULMONARY VASCULAR PERMEABILITY
these other data are not necessarily inconsistent with ours. In addition, because the DTPA method depends on aerosol droplets for tracer delivery and the [68Ga]transferrin tracer is bloodborne, we cannot exclude the possibility that cigarette smoke has an effect on central airway epithelium that is not evident when endothelial permeability is measured in the lung periphery. For example, DTPA clearance and non-PET protein flux measurements have been compared in normal controls, patients with ARDS, and subjects with cigarette exposure (38). Unlike our data, which can clearly distinguish ARDS patients from normal smoking subjects, DTPA clearance wasequally abnormal during ARDS and after cigarette exposure. We found no difference between PTCER values for smokers and nonsmoking normal subjects. Based on the same sample size and coefficient of variation reported in this study,the minimum detectable difference between groups with a power of 0.75 would be 18 x 1O-4/min. Thus, aithough we cannot exclude a very small difference between PTCER values for smokers and nonsmoking normal subjects, any such difference would not complicate the interpretation of PTCER values in patient groups like those already reported (8, 9, 39). References 1. Holden WE, Maier JM, Malinow MR. Cigarette smoke extract increases albumin flux across pulmonary endothelium in vitro. J Appl Physiol 1989; 66:443-9. 2. Todisco T, Dottorini M, Rossi F, Baldoncini A, Palumbo R. Normal reference values for regional pulmonary airspace epithelial permeability. Respiration 1989; 55:84-93. 3. Rinderknecht J, Shapiro L, Krauthammer M, et al. Accelerated clearance of small solutes from the lungs in interstitial lung disease. Am Rev Respir Dis 1980; 121:105-17. 4. Rinderknecht J, Krauthammer M, Uszler JM, Toplin G, Effros RM. Solute transfer across the alveolar capillary membrane in pulmonary fibrosis. Am Rev Respir Dis 1977; 115:156. 5. Chopra SK, Taplin GV, Tashkin DP, Elan D. Lung clearance of soluble radioaerosols of different molecular weights in systemic sclerosis. Thorax 1979; 34:63-7. 6. Mason G, Uszler JM, Effros RM. Differentiation between hemodynamic and nonhemodynam-
ic pulmonary edema by a scanning procedure. Am Rev Respir Dis 1981; 123:238. 7. Mintun MA, Dennis DR, Welch MJ, Mathias CJ, Schuster DP. Measurements of pulmonary vascular permeability with PET and gallium-68 transferrin. J Nucl Med 1987; 28:1704-16. 8. Calandrino FS, Anderson DJ, Mintun MA, Schuster DP. Pulmonary vascular permeability during the adult respiratory distress syndrome: a positron emission tomographic study. Am Rev Respir Dis 1988; 138:421-8. 9. Kaplan JD, Calandrino FS, Schuster DP. Abnormal pulmonary vascular permeability in radiographically normal lung in patients with focal pneumonia. Am Rev Respir Dis 1991; 143:150-4. 10. Velazquez M, Weibel ER, Kuhn C, Schuster DP. PET evaluation of pulmonary vascular permeability: a structure-function correlation. J Appl Physiol 1991; 70:2206-16. 11. Mintun MA, Warfel TE, Schuster DP. Evaluating pulmonary vascular permeability with radiolabeled proteins: an error analysis. J Appl Physiol 1990; 68:1696-706. 12. Schuster DP. Positron emission tomography: theory and its application to the study of lung disease. Am Rev Respir Dis 1989; 139:818-40. 13. Ter-Pogossian MM, Ficke DC, Yamomoto M, Hood JT. Design characteristics and preliminary testing of SUPERPETT I, a positron emission tomograph utilizing photon time-of-flight information (lOF-PET). IEEE Trans Med Imag 1982; 1:37-41. 14. Moerlein SM, Welch MJ. The chemistry of gallium and indium as related to radiopharmaceutical production. Int J Nucl Med BioI /981; 8:277-87. 15. Altman PL, Dittmer DS, eds. Respiration and circulation. Bethesda, MD: FASEB 1971; 25. 16. Effros RM, Mason GR, Mena I.••mTc-DTPA aerosol deposition and clearance in COPD, interstitial disease and smokers. J Thorac Imaging 1986; 1:54-60. 17. Bylock A, Bondjers G, Jansson I, Hansson HA. Surface ultrastructure of human arteries with special reference to the effects of smoking. Acta Pathol Microbiol Scand 1979; 87:201A-9A. 18. Minty BD, Royston D, Jones JG, Hulands GR. The effect of nicotine on pulmonary epithelial permeability in man. Chest 1984; 86:72-4. 19. Allen DR, Browse NL, Rutt DL, Butler L, Fletcher C. The effects of cigarette smoke, nicotine and carbon monoxide on the permeability of the arterial wall. J Vasc Surg 1988; 7:139-52. 20. Staub NC, Hyde RW, Crandall E. Workshop on techniques to evaluate lung alveolar-microvascular injury. Am Rev Respir Dis 1990; 141: 1071-7. 21. MacNee W, Wiggs B, Belzberg AS, Hogg JC. The effect of cigarette smoking on neutrophil kinetics in human lungs. N Eng! J Med 1989; 321:924-8. 22. Hunninghake GW, Crystal RG. Cigarette smoking and lung destruction: accumulation of neutrophils in the lungs of cigarette smokers. Am Rev Respir Dis 1983; 128:833-8. 23. Ludwig PW, Schwartz BA, Hoidal JR, Niewoehner DE. Cigarette smoking causes accumula-
715 tion of polymorphonuclear leukocytes in alveolar septum. Am Rev Respir Dis 1985; 131:828-30. 24. Wollmer P, Albrechtsson U, Brauer K, Eriksson L, Jonson B, Tylen U. Measurement of pulmonary density by means of x-ray computerized tomography. Chest 1986; 90:387-91. 25. Spain DM, Siegel H, Bradess VA. Emphysema in apparently healthy adults. JAMA 1975; 224:322-5. 26. Niewoehner DE, Kleinerman J, Rice DB. Pathologic changes in the peripheral airways of young cigarette smokers. N Eng! J Med 1974; 291:755-8. 27. Brudin LH, Rhodes CG, Valind SO, Wollmer P, Hughes JMB. Regional lung density and blood volume in nonsmoking and smoking subjects measured by PET. J Appl Physiol 1987; 63:1324-34. 28. Schuster DP, Mintun MA, Green MA, lerPogossian MM. Regional lung water and hematocrit determined by positron emission tomography. J Appl Physiol 1985; 59:860-8. 29. Brudin LH, Valind SO, Rhodes CG, Turton DR, Hughes JMB. Regional lung hematocrit in humans using positron emission tomography. J Appl Physiol 1986; 60:1155-63. 30. Albelda SM, Sampson PM, Haselton FR, et al. Permeability characteristics of cultured endothelial cell monolayers. J Appl Physiol 1988; 64:308-22. 31. Gorin AB, Steward PA. Differential permeability of endothelial and epithelial barriers to albumin flux. J Appl Physiol 1979; 47:1315-24. 32. Simani AS, Inoue S. Hogg JC. Penetration of respiratory epithelium of guinea pigs following exposure to cigarette smoke. Lab Invest 1974; 31:75-81. 33. Burns AR, Hosford SP, Dunn LA, Walker DC, Hogg JC. Respiratory epithelial permeability after cigarette smoke exposure in guinea pigs. J Appl Physiol 1989; 66:2109-16. 34. Effros RM, Mason GR. Measurements of pulmonary epithelial permeability in vivo. Am Rev Respir Dis 1983; 127:559-65. 35. Jones JG, Lawler P, Crawley JCW, Minty BD, Hulands G, VeaHN. Increased alveolar epithelial permeability in cigarette smokers. Lancet 1980; 1:66-8. 36. Minty BD, Jordan C, Jones JG. Rapid improvement in abnormal pulmonary epithelial permeability after stopping cigarettes. Br Med J 1981; 282:1183-6. 37. Mason GR, Uszler JM, Effros RM, Reid E. Rapidly reversible alteration of pulmonary epithelial permeability induced by smoking. Chest 1983; 83:6-11. 38. Braude S, Nolop KB, Hughes JMB, Barnes PJ, Royston D. Comparison of lung vascular and epithelial permeability indices in the adult respiratory distress syndrome. Am Rev Respir Dis 1986; 133:1002-65. 39. Kaplan JD, Trulock EP, Kaiser LR, Cooper JD, Schuster DP, Washington University Lung Transplant Group. Pulmonary vascular permeability changes during rejection after lung transplantation. Am Rev Respir Dis 1989; 141:A683.
JAMES D. KAPLAN, FRANK S. CALANDRINO, and DANIEL P. SCHUSTER4
Introduction
Several studies suggest that cigarette smoke can affect pulmonary vascular permeability. For example, cigarette smoke increases permeability to albumin flux in an endothelial cell monolayer (1). Epithelial permeability is also increased in chronic smokers as measured in vivo by clearance rates of [99mTc]-diethylenetriamine pentaacetic acid (DfPA) aerosol (2). These findings present a potential drawback to studying certain forms oflung disease because the specificity of an abnormal result is reduced if the subject smokes cigarettes. Nevertheless, epithelial permeability measurements by the 99mTc-DTPA clearance technique have been used to assess acute and chronic lung diseases, including pneumoconioses (3), idiopathic pulmonary fibrosis (4), inflammatory interstitial lung disease(5), and adult respiratory distress syndrome (ARDS) (6). The overlap in values between otherwise normal smokers and subjects with lung disease makes it difficult to interpret "abnormal" epithelial permeability in some patients. In previous studies with positron emission tomography (PET), we described a method to evaluate pulmonary vascular permeability by measuring the pulmonary transcapillary escape rate (PTCER) for radiolabeled transferrin. This noninvasive, repeatable method has been used to study different forms of acute lung injury, including experimental oleic acid lung injury (7), (ARDS) (8), and acute pneumonia (9). In experimental lung injury, abnormalities in PTCER correlate with histologic evidence of both endothelial and epithelial damage (10). However, if the PET technique could not adequately distinguish between abnormalities induced by smoking alone from those associated with ARDS, as is the case for the epithelial permeability measurement, its usefulness would clearly be reduced. Therefore, in the present study, we evaluated pulmonary vascular 712
SUMMARY Positron emission tomography (PET) can be used to evaluate pulmonary vascular endothelial permeability by measuring the pulmonary transcapiliary escape rate (PTCER) for radlolabeled transferrin. Because epithelial permeability, as evaluated by other techniques, Is significantly affected by cigarette smoking, we used PET to compare the effects of smoking on extravascular lung density (EVD)and PTCERin seven normal chronic cigarette smokers within 30 min of smoking a cigarette and seven normal nonsmokers. We found no difference in PTCERand EVD between the two groups. We conclude that the Interpretation of acute or chronic lung InJury studies with PET should not be affected by cigarette smoking In the subject population. AM REV RESPIR DIS 1992; 145:712-715
permeability in chronic smokers by measuring PTCER and compared these values with nonsmoking volunteer subjects with no evidence of lung disease. Methods Subjects We studied seven healthy nonsmoking adult volunteers and seven chronic cigarette smokers. Subjects were between 18 and 50 yr of age and were of either sex. Normal volunteers had no evidence by history or physical examination of active or prior cardiopulmonary disease. Smokers had smoked 10to 40 cigarettes daily for 3 to 15 pack-years and were asked to smoke a cigarette within 30 min of beginning the PET study. Despite their chronic cigarette use, smokers had no evidence of cardiopulmonary diseaseby medical history or physical examination.
Study Protocol The theory and methods of PET measurements have been reviewed recently (11, 12). PET scan data were acquired on the SUPERPETT I body scanner (13) and displayed as seven transaxial tomographic slices with a center-to-center separation of 15 mm and an intrinsic in-plant resolution of 18 mm. Subjects were studied in a supine position. A PET study involved collecting a background scan, a transmission scan, and a series of emission scans. The background and transmission scans wereperformed to correct emission scans for errors in the measurement of tissue activity introduced by tissue attenuation. Because activity attenuation is proportional to tissue density, the transmission scan also provided a density image resembling a
standard X-ray computed tomography image, which was used to define anatomic regions of interest. Serial emission scans were then collected for 60 min beginning 2 min after injection of up to 6 mCi of [68Gajcitrate, prepared according to previously published methods (7, 14, 15). Time-activity data were obtained for each region of interest in the lung and within the cardiac blood pool on each emission scan. These data were decay corrected to the time of tracer injection. The blood pool activity was analyzed by two sequential linear regressions to produce a biexponential expression for the fitted blood curve as previously described (7). The tissue and blood time-activitydata were analyzed with a two-compartment mathematical model. Using this model, the vascular-toextravascular rate constant k, was calculated. Since protein flux is affected by surface
(Received in original form February 11, 1991 and in revised form May 20" 1991) 1 From the Respiratory and Critical Care Medicine Division, Department of Internal Medicine, Washington University Medical School, St. Louis, Missouri. 2 Supported by Grant No. POI HL 13851 from the National Institutes of Health and Grant No. DE-FG02-87ER60512 from the Department of Energy. 3 Correspondence and requests for reprints should be addressed to Daniel Schuster, M.D., Respiratory and Critical Care Medicine Division, Box 8052, Washington University School of Medicine, St. Louis, MO 63110. 4 Established Investigator of the American Heart Association and Career Investigator of the American Lung Association.
713
PET STUDY OF SMOKING AND PULMONARY VASCULAR PERMEABILITY
Fig. 1. Single-slice images of total density, blood volume, extravascular density, and permeability from a representative study of a normal cigarette smoker. Lung regions and mean values for each lung are identified on each image.
area available for exchange, k, was normalized to regional blood volume, which is assumed to be proportional to this surface area. Regional plasma volume was determined by calculating the ratio of PET activity during the initial 2 min of data collection, when virtuallyall [68Ga]transferrin plasma label is still intravascular (7), to the corresponding integral of the fitted blood curve. Blood volume was determined from the plasma volume measurement, adjusting for hematocrit as measured by the capillary tube centrifuge method. PTCER wasthen calculated as k,/regional blood volume. The attenuation scan was scaled to the known density of blood so that the cardiac blood pool had a density of 1.05 g/ml (IS). This resulted in an image of regional lung density (rLD), including both the vascular and extravascular compartments. Regional extravascular density (EVD) was then calculated as rLD minus regional intravascular density.
PET measurements were expressed in the following units: EVD (g/IOO mllung), rLD (g/IOO ml lung), and PTCER (l0-4 mirr").
Image Analysis Lung regions were identified on trans axial slicesof the transmission scan for each subect, stored in computer memory, and then superimposed on images ofrLD, blood volume, EVD, and PTCER (figure I). Regions of interest included the entirety of the right and left lungs on all slices above the diaphragm, representing approximately 10.5 em of lung in a craniocaudal direction. Statistical Analysis The data are expressed as the mean ± SD. Analysis of variance (ANOVA) techniques were used to test for significant differences (p < 0.05) among the mean values. Within the ANOVA, t tests were conducted among previously chosen pairs of means to establish statistically significant differences. A
least-squares means analysis by the general linear model procedure of the StatisticalAnalysis System (SAS Institute, Cary, NC) for the IBM-PC was used for these statistical calculations.
Results
Mean age for nonsmoking and smoking subjects was not different (28.6 ± 8.3versus 30.4 ± 9.9, respectively, p = NS). Mean PTCER, rLO, and EVO were not different between smokers and nonsmokers (p = NS, figure 2). A small but statistically significant difference in rLO was present between the left and right sides within each group (nonsmokers p < 0.0025, smokers p < 0.0321,table 1). In addition, the nonsmokers' right lung rLO showed a small but statistically significant difference from the rLO of either lung in the smokers (p < 0.0025).
714
KAPLAN, CALANORINO, AND SCHUSTER
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ing due to the position of the heart in the left hemithorax. There were also small density differences between the lowest value for nonsmokers (the right lung) and both the right and left lung rLD values for smokers. Since rLD is the sum of intravascular density plus EVD and EVD was not different between the two groups, this rLD increase is a reflection of an increased pulmonary capillary blood volume in the smokers 30 min after a cigarette. Because the bilaterally increased rLD in smokers was only statistically significant when compared with the least dense lung in nonsmokers, it is ofuncertain physiologic importance. Nevertheless, other studies have suggested that an increased density is plausible after cigarette smoking. For instance, nicotine is a well-known vasodilator and may be expected to cause increased regional blood volumes (18, 19). Studies of labeled erythrocytes and neutrophils demonstrate neutrophil retention in smokers' lungs (20, 21), and neutrophils and other cells are increased in lung capillaries for up to several days after exposure to cigarette smoke (21, 22). Our findings are also consistent with standard radiographic computed tomographic density measurements in smokers and nonsmokers (23, 24). On the other hand, our data are not explained by the peribronchial inflammation that can be present histologically in some chronic smokers (25). Changes due to inflammation, which are "ex-
The greatest magnitude of this difference was only 17"10 between this value for nonsmokers' right lung rLD and the densest average value (the smokers' left lung). No difference in PTCER or EVD was present between smokers and nonsmokers or between left and right sides within groups (table 1). Discussion
To determine the effect of cigarette smoking on the PTCER measurement as measured by PET, we evaluated endothelial permeability in otherwise normal smokers. The data are important because (1) permeability estimates by techniques that measure epithelial injury have been abnormal in otherwise normal smokers (16); (2) in vitro studies suggest that smoke causes abnormal permeability in endothelial tisue (1, 17); and (3) studies of subjects with acute or chronic lung disease frequently include smokers who may bias the results if smoking itself contributes to abnormal values of "permeability" (18). In this study, we found a small difference between the right and left lung total density in both smokers and nonsmokers. BecausePET data are expressed in terms of unit volume, regions of normal tissue can have higher density values simply as a result of atelectasis. Therefore, this biologically small elevation in rLD (up to 17%) on the left side in this study may reflect in part anatomic crowd-
TABLE 1 MEAN VALUES FOR NONSMOKERS ANO SMOKERS' EVO (g/100 ml)
rlO (g/100 ml)
Nonsmokers Smokers All subjects
Right
left
Mean
Right
left
35 ± 5t; 40 ± 5t 38 ± 4t
39 ± 6 42 ± 4 40 ± 5
37 ± 5 41 ± 4 39 ± 5
19 ± 5 19 ± 4 19 ± 4
17 ± 3 17 ± 4 17 ± 4
Mean
18 ± 4 18 ± 4 18 ± 4
PTCER (10-' min-f) Right
left
Mean
20 ± 12 22 ± 15 21 ± 9 15 ± 13 26 ± 16 21 ± 14 18 ± 13 24 ± 15 21 ± 11
• Mean ± SO values lor regional lung density (rLO), extravascular density (EVD), and permeability (PTCER) lor nonsmokers, smokers, and all subjects evaluated. t p < 0.05 right versus left. :; p < 0.05 lor right lung nonsmokers versus either lung smokers rLO values.
travascular," should be manifest as both increased EVD and rLD. Inflammation may be more relevant in patients with active chronic bronchitis (26), a group excluded by definition from this study. One other study used PET to measure density in normal smokers and nonsmokers (27). In a single tomographic slice at the midthoracic level (as opposed to an average from multiple contiguous slices in our study), EVD was 16% higher for smokers. Consistent with our data, total density was slightly greater in the left lung compared with the right lung, although this difference was not statistically significant. These differences between our data and this other study may be related to differences in the study populations, in the methods for defining regions of interest, or in the method for measuring blood volume. Although our own data in dogs suggest that 68Ga-Iabeled transferrin and CIsO-Iabeled red blood cells measure blood volume equally well (28), data in humans suggest that small differences between the two methods can occur (29). Overall, the magnitude of the density differences in either study, although sometimes statistically significant, were quite small. Because permeability measurements werenot performed in this other PET study, it is difficult to evaluate the importance of these differences in EVD. Some investigators have found the permeability to labeled albumin to be abnormal in endothelial cell monolayers exposed to cigarette smoke (1). It is difficult to compare our data with this in vitro effect. The in vivo dose of inhaled smoke may have been less than that used in vitro, or the levelof injury detected in vitro may be below the level of detection of our method. Moreover, direct comparisons suggest that there may be important differences between the permeability characteristics of endothelial cell monolayers and blood vessels in vivo (30) and between the permeability characteristics of endothelial and epithelial cell barriers as well (31). It is possible that an epithelial injury can bel detected by one method while the endothelial barrier remains intact. This would be consistent with the histologic effects of modest smoke exposure in guinea pigs, in which there is damage to epithelial but not endothelial structures (32, 33). Abnormalities are detectable in smokers for at least 7 days after smoking cessation by epithelial permeability techniques (34-37). Since epithelial permeability studies do not measure pulmonlary vascular endothelial injury per se,
PET STUDY OF SMOKING AND PULMONARY VASCULAR PERMEABILITY
these other data are not necessarily inconsistent with ours. In addition, because the DTPA method depends on aerosol droplets for tracer delivery and the [68Ga]transferrin tracer is bloodborne, we cannot exclude the possibility that cigarette smoke has an effect on central airway epithelium that is not evident when endothelial permeability is measured in the lung periphery. For example, DTPA clearance and non-PET protein flux measurements have been compared in normal controls, patients with ARDS, and subjects with cigarette exposure (38). Unlike our data, which can clearly distinguish ARDS patients from normal smoking subjects, DTPA clearance wasequally abnormal during ARDS and after cigarette exposure. We found no difference between PTCER values for smokers and nonsmoking normal subjects. Based on the same sample size and coefficient of variation reported in this study,the minimum detectable difference between groups with a power of 0.75 would be 18 x 1O-4/min. Thus, aithough we cannot exclude a very small difference between PTCER values for smokers and nonsmoking normal subjects, any such difference would not complicate the interpretation of PTCER values in patient groups like those already reported (8, 9, 39). References 1. Holden WE, Maier JM, Malinow MR. Cigarette smoke extract increases albumin flux across pulmonary endothelium in vitro. J Appl Physiol 1989; 66:443-9. 2. Todisco T, Dottorini M, Rossi F, Baldoncini A, Palumbo R. Normal reference values for regional pulmonary airspace epithelial permeability. Respiration 1989; 55:84-93. 3. Rinderknecht J, Shapiro L, Krauthammer M, et al. Accelerated clearance of small solutes from the lungs in interstitial lung disease. Am Rev Respir Dis 1980; 121:105-17. 4. Rinderknecht J, Krauthammer M, Uszler JM, Toplin G, Effros RM. Solute transfer across the alveolar capillary membrane in pulmonary fibrosis. Am Rev Respir Dis 1977; 115:156. 5. Chopra SK, Taplin GV, Tashkin DP, Elan D. Lung clearance of soluble radioaerosols of different molecular weights in systemic sclerosis. Thorax 1979; 34:63-7. 6. Mason G, Uszler JM, Effros RM. Differentiation between hemodynamic and nonhemodynam-
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