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Markers of Physiologic Effects in Intact Organisms This section discusses markers of pul- monary response that can be applied in studies of intact human subjects (all can also be applied to animals). The focus is on physiologic tests of respiratory system function that reflect early biologic ef- fects or underlying changes in lung struc- ture, function, or sensitivity to inhaled materials. Most of the techniques reviewed here can be applied in a relatively noninvasive manner and do not require anesthesia, cath- eterization, or collection of tissue sam- ples. Many are suitable for epidemiologic studies of large populations and have the characteristics of mobility of equipment, short subject interaction time, minor requirement for subject training, ability to be performed by technicians, and auto- mated data processing; an example of such a technique is spirometry, the most common of those discussed in this section. The equipment, personnel, and sample- collection requirements of others imply that they are likely to be used only in sta- tionary facilities. Many centers are suit- able for these tests, but they are most appropriate for evaluation of small popu- lations. Techniques that use gamma-camera imaging are examples of this class because gamma-camera equipment is large. Other 43 approaches require equipment and exper- tise that are likely to be present only in a few specialized laboratories, for ex- ample, measurements of the dispersion or clearance of inhaled boluses of particles. The markers of early biologic effects or altered structure/function discussed here are in four general categories. The first includes markers of respiratory or gas-exchange function of the lung. These are derived from tests of ventilation and its control, lung mechanical properties, intrapulmonary gas distribution, and alveolar-capillary gas exchange. A wide variety of such tests have been developed and are in common use to evaluate lung func- tional competence. Although all these tests yield markers of lung response, only a few thought most likely to have potential for detecting responses of populations to environmental exposures are discussed in detail. The second category includes markers of increased airway reactivity, both to specific environmental agents and to standardized physical or pharmacologic bronchial provocation. Although these are usually indexes of respiratory func- tion, they are distinct from the first category, in that the focus is not on gas exchange. The third category includes markers derived from measurement of the .. . . ~

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44 clearance of particles. These measure- ments examine an important set of defense mechanisms of the respiratory system, and alterations of the mechanisms sometimes give an early indication of an adverse impact of inhaled environmental agents. The fourth category includes markers of increased permeability of the air-blood barrier, which is sometimes an early feature of lung injury due to inhaled materials. The usefulness of assays of physiologic function is generally limited to their ability to demonstrate responses to in- haled environmental agents. They reveal the functional manifestations of struc- tural changes in the respiratory system, whether the changes are transient (e.g., bronchoconstriction) or lasting (e.g., fibrosis). As a group, the assays have little specificity for specific environ- mental agents. The lung responds to injury in a limited number of ways, and the func- tional manifestation of a given type of injury is often the same, regardless of the causative agent. An exception to both those generalizations might be the useful- ness of an increase in airway sensitivity to specific agents as a marker of exposure and sensitization. The sensitivity of physiologic tests varies, but many demonstrate substantial intersubject variability. The sensitivi- ty is often improved when control popula- tions are studied or when individual base- line values are obtained; however, there is often a reliance on predicted normal values. The results of physiologic assays generally depend on technique, and both the degree of standardization of technique and the magnitude of the data base for pre- dicting normal values vary widely from assay to assay. Some of the physiologic tests offer the advantage that a response, once detected, can be placed in a context that can be interpreted in terms of its practical impact on the subject. That is based on knowledge of correlations among measured values related to function, sub- jective perception of ill health, clinical lung disease, and impairment of physical performance. MARKERS IN PULMONARY TOXICOLOGY RESPIRATORY FUNCTION The respiratory functions of the lung include mechanisms involved in ventila- tion, gas distribution,alveolar-capil- lary gas exchange, and perfusion. Although developments in the understanding and evaluation of respiratory function are continuing, most of these physiologic phenomena are well-studied, and many as- says of function are long established. Table 3-1 lists several common assays of respiratory function. Note that each list- ed category contains numerous individual tests or measured characteristics. Only a few of the many assays of respira- tory function are discussed in detail in this section, but all the assays listed are useful in a clinical setting and yield potential markers of response to environ- mental exposures. As there is considerable literature on the performance and inter- pretation of the assays; it would be inap- propriate to review it all here. The intent of this review is to comment on the function tests that the Committee thought were most likely to have potential for studies of the effects of environmental exposures in occupational groups or more general populations. Considerable attention has been focused on the development of respiratory function tests that are sensitive to alterations in the region of terminal bronchioles and respiratory bronchioles-i.e., small- airway disease. That anatomic region is at particular risk from several types of inhaled toxicants. Therefore, respira- tory function tests that have been proved (or proposed) to have particular sensi- tivity to small-airway disease are dis- cussed here. Current knowledge suggests that a given test has little usefulness for producing markers of effects of speci- fic environmental exposures. Spirometry The forced expiratory maneuver, which records the time taken to expel as quickly as possible as much gas as possible from a full deep inspiration, is the mainstay of both clinical pulmonary physiology and epidemiologic field studies designed to

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PHYSIOLOGYIN INTACT ORGANISMS 45 TABLE 3-1 Assays of Respiratory Function Breathing pattern Respiratory frequency, tidal volume, and minute volume Inspiratory-expiratory times and flow rates Alveolar ventilation Physiologic subdivisions of lung volume Total lung capacity Vital capacity Functional residual capacity Residual volume Inspirato~y and expiratory resents volumes Spirometry (forced exhalations Forced vital capacity (FVC) Forced expired volume in 1 second (FEV') and %FVC in 1 second Peak aspiratory flow Mean midexpiratory flow Flows at selected absolute lung volumes or portions of FVC Breathing mechanics Dynamic lung mechanics Dynamic lung compliancea Total pulmonary resistance Airway resistance and conductance, and specific airway resistance and conductance Oscillation mechanics (respiratory system impedance, composed of compliance, resistance, and inertanceJa Static-quasistatic lung compliancea Intrapulmona~y gas distribution Single-breath gas washouts Multiple-breath gas washout Imaging of radiolabeled gas and particlesa Particle bolus distributions Alveolar-capillary gas transfer Blood gases, pH, and alveolar-arterial gas tension differences Oxygen and carbon dioxide exchange at rest Diffusing capacity for carbon monoxides Gas exchange during exercises Multiple-gas evaluation of ventilation-perfusion relationships Evaluation of respiratory control . aDiscussed In this report. explore the development of chronic ob- structive pulmonary disease. The volume of air expelled in the first second is termed the forced expiratory volume in 1 second (FEVER. The total amount expelled is called the forced vital capacity (FVC). The flow-volume curve is a plot of expira- tory flow rate against expired volume, and it is also analyzed to evaluate flow limitation. Patients with chronic obstructive res- piratory diseases are those whose airflow limitation prevents them from continuing activities that they would otherwise be able to perform (Speizer and Tager, 1979~. There is a continuum between the normal state and the diseased state. Most people would consider themselves substantially disabled when their FEV, approached 40- 50% of the predicted value. Most physi- cians would take an FEV1 below 65% of the predicted value as indicative of obstruc- tive disease. LEVI tends to decline smoothly in adult life with modest acceleration with in- creased age (Figure 3-1 ) (Speizer and Tager, 1979~. Several studies have shown that airflow function is a strong predictor of morbidity (Fletcher et al., 1976~. Thus, FEVER is a good biologic marker of risk of developing obstructive pulmonary disease.

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46 FIGURE 3-1 Decline of FEVER at normal rate (solid line) and at accelerated rate (dashed line). "A" represents a person who has attained a "normal" maximal F~V~ during lung growth and development. "B" represents a person whose mammal FEVER has been reduced by childhood respiratory infection (CAO). Repnnted with permission from Samet et al., 1983. Additional measures of ventilatory function that are general biologic markers of risk of chronic respiratory disease are based on a forced expiration. To use them, one must have recording devices that capture the essential components of the maneuver or the entire curve. The total volume of an expiration, the FVC, has been recognized since the mid-nineteenth cen- tury as a predictor of "vital status" (Hut- chinson, 1846~. In the 1960s, the Framing- ham heart program found FVC to be a predic- tor of total mortality (Kennel et al., 1980~. More recently, FEW and FVC measure- ments obtained some 20 years previously were used to predict specific respiratory disease mortality (Peso et al., 1983~. Other components that are believed to rep- resent the flow characteristics of the airways include flow after 50 or 75% of the volume has been forcibly expelled (forced expiratory flow of 50 or 75%, FEF50 or FEF75) and the maximal midexpiratory flow (MMEF, the slope of the line drawn between points at 25 and 75% of the FVC). The same measures have all been used (with other measures discussed below) to test the effects of exposure to an array of environmental agents, generally at concentrations that exceed only slightly those occurring in the environment. In each case, pre-exposure, postexposure, and recovery measurements were compared. The interpretation of those measures as biologic markers of risk is only partly MARKERS IN PULMONARY TOXICOLOGY 5 4 2 1 o B ~ Normal Normal ~ CAO 25 35 45 55 YEARS 65 75 understood. When asthmatic subjects are exposed at rest to SO2 at 0.25-0.5 ppm, some undergo significant reductions in FIVE (Sheppard et al., 1980~. Whether the re- sults predict which asthmatics are more susceptible to naturally occurring en- vironmental insults is as yet unknown. However, we do know that children who re- port wheezing or asthma generally have more respiratory symptoms than those who do not when exposed to ambient environments with particulate pollution (Ware et al., 1984~. No relation between pollutant con- centration and magnitude of FEV, FVC, or MMEF change has been discerned, but chil- dren with a history of wheezing clearly have lower MMEF. In most chronic respiratory diseases other than asthma, lost ventilatory func- tion does not return. By the time impair- ment is judged to be significant, there is little need for subtle biologic markers of risk of disease (it might still be ap- propriate to use markers to study mechan- isms). Therefore, it is important to con- sider how the sensitivities of the other markers compare with the sensitivity of FEY,. For example, subtle decreases in flow at 50 or 75% of vital capacity (FEF50 or FEF75) might be associated with cigar- ette-smoking without necessarily being simultaneously associated with reduced FEY,. Some have argued that those findings represent changes in small airways (Gelb and Zamel, 1973~. Typically, reduced FEF50

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PHYSIOLOGYIN INTACT ORGANISMS or FEF75 without a reduced FEVER has not been used to define obstructive airway disease (Speizer and Tager, 1979~. MMEF similarly might be reduced in association with ex- posure to respiratory irritants, but this has not typically been used to define ob- structive disease without a reduced FEW. Those more subtle measures are useful as biologic markers, because they might indi- cate earlier or more subtle damage to small airways that, if not reversed, could lead to more severe and irreversible damage reflected in reduction in LEVI and even- tually in FVC. Mechanical Properties of the Lung Dynamic Lung Mechanics Measurement of dynamic lung mechanics is a means of assessing the work of breath- ing. The work of breathing is incurred in the need to overcome elastic, resistive, and inertial forces of the lung tissue and air column (Mead and Agostoni, 1964; Rodarte and Rehder, 1986~. Dynamic lung mechanics-lung mechanical properties during breathing-are usually expressed in terms of dynamic lung compliance (indicative of work required to stretch the lung) and airway resistance (indic- ative of work required to overcome resis- tance to airflow). Classical measures of dynamic lung mechanics are often useful in evaluating clinical lung disease, but by themselves have low potential as sensi- tive markers of lung response to environ- mental exposures. Tests commonly applied in epidemiologic studies to detect abnormalities of respir- atorY function typically evaluate lung volume and resistance to airflow, but do not examine the compliance. Lung compli- ance is reduced in disorders such as in- flammation and fibrosis, which increase lung elastic recoil, and is increased in diseases like emphysema, which decrease elastic recoil. Measurement of static or quasistatic compliance demonstrates those changes more sensitively than does measurement of dynamic compliance. Aside from forced-oscillation methods, measure- ment of dynamic compliance requires place- ment of an esophageal balloon catheter. 47 Although that is not difficult or hazar- dous, it is sufficiently time-consuming and unpleasant for its use to be limited usually to selected clinical subjects and to the physiology laboratory. Oscillation techniques now constitute the most likely use of lung compliance as a marker of response. Dynamic lung compliance depends on breathing frequency. Compliance de- creases with increasing breathing fre- quency, because of regional inhomogen- eities among lung units (Otis et al., 1956~. Measurement of the frequency de- pendence of compliance was introduced as one of the first tests "specific" for small-airway disease (Woolcock et al., 1969~. Compliance is measured as the sub- ject breathes over a range of frequencies, and the magnitude of the reduction is noted. Although the dependence of compli- ance (or resistance) on frequency is often mentioned as a potential marker of small- airway disease, these changes have actual- ly been correlated with structural changes in the lung in only a few studies (Berend, 1982~. The dependence on frequency has been shown to be abnormal in a large portion of asymptomatic young smokers (Martin et al., 1975~. The compliance test is not broadly used, and there are few data on which to base either an estimate of its usefulness in population studies or es- timates of normal values. The most common method of assessing re- sistance to airflow is spirometry during forced exhalation. Resistance to airflow during either forced exhalation or tidal breathing is commonly used to indicate response in studies of airway sensitivity and in evaluating experimental exposures of humans to inhaled toxicants. Although resistance can be measured during tidal breathing with esophageal catheters or oscillation methods, it is most commonly measured with plethysmography (Leith and Mead, 1974; Zarins and Clausen, 1982~. The subject is seated within a body pleth- ysmograph (a box with transducers that sense changes in pressure) and breathes with a panting pattern while flow at the mouth and pressure changes within the plethysmograph are measured. The airway is then occluded, and mouth pressure is

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48 measured as representative of alveolar pressure. The resulting data are used to calculate resistance and thoracic gas volume. Resistance can depend on volume, so it is often divided by volume and ex- pressed as specific airway resistance (resistance per unit volume) or its recip- rocal, specific airway conductance. Current measurements of dynamic lung mechanics are useful clinical tools, but are unlikely to gain substantially broader use in population studies. The information obtained represents the integrated re- sponse of the entire lung; the lack of re- gional specificity and the lack of sensi- tivity due to intersubject variability reduce its utility as a marker of subtle effects. The greatest potential for devel- opment as a marker appears to lie in the use of oscillation methods because they are noninvasive and provide considerable information without requiring difficult procedures as described below. Respiratory System Impedance (Oscillation MechanicsJ Measurement of "oscillation mechanics" is a means of evaluating the mechanical properties of the respiratory system. The technique provides a marker of response in the form of information on changes in the mechanical properties of the lung. The technique is rapid and and requires little cooperation from the subject-char- acteristics that make it suitable for epi- demiologic studies. It provides indexes of lung compliance, as well as resistance; thus, it has potential for adding to the spectrum of information obtained in popu- lation studies. Tests of oscillation me- chanics are in use for measuring the in- tegrated compliance and resistance of an entire lung, but its potential as a marker is primarily in describing mechanical properties of specific regions of the res- piratory system. The extent of the poten- tial is uncertain and is the focus of cur- rent developmental work. Oscillation mechanics was recently reviewed by Peslin and Fredberg (1986~. The general approach is to superimpose an oscillating pressure signal on the air- way during normal tidal breathing with MARKERS IN PULMONARY TOXICOLOGY a loudspeaker or pump. The frequency of oscillation is higher than the respiratory frequency of the subject, and the oscillat- ing pressure and flow changes are small. The resulting pressure, volume, or flow perturbations in the air column are meas- ured and used to calculate values of com- oonents of the mechanical impedance (re- s~stance, compliance, and inertance) of the respiratory system. The response of the respiratory system to an oscillating signal is determined by its impedance, which in turn is deter- mined by its anatomic and mechanical prop- erties. The overall response of the system represents an integration of the elastic, resistive, and inertial charac- teristics of each component of the system. In the simplest form of the assay, oscilla- tion at a single frequency is used to meas- ure dynamic compliance and airway resis- tance of the entire lung, without the need for a body plethysmograph or esophageal catheter. By manipulating the oscillating signal and analyzing the resulting re- sponse, one can theoretically extract information specific for different mech- anical properties and for different ana- tomic structures. Two approaches have been used for inter- preting respiratory system impedance. One is the empirical association of changes in impedance with lung abnormalities (Kejeldgaard et al., 1976~. The second is the estimation of specific impedance parameters by fitting impedance data to mathematical models of the respiratory system, which are based on mechanical or electric analogues (Jackson et al., 1984; Peslin et al., 1986~. The latter approach should provide more descriptive informa- tion, if model parameters can be correlated with physiologic elements of the respira- tory system. Much of the effort in this field is di- rected toward development of improved models of respiratory system impedance. Previous work focused primarily on oscil- lating frequencies of 2-32 Hz, and it now appears that such data allow reliable ex- traction of only the integrated compli- ance, resistance, and inertance of the total respiratory system (Jackson et al., 1984~. By extending the range of oscillat

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PHYSIOLOGYIN INTACT ORGANISMS ing frequency, one can obtain statistical- ly reliable estimates of additional param- eters. For example, Jackson and Watson ( 1982) differentiated between central and peripheral resistance, compliance, and inertance by fitting oscillation data from rats to a six-parameter model. Their group has obtained similar results with other animal species, but has encountered difficulties in applying such models to data from humans. They hypothesize that models for humans need to account for shunting of flow in upper airways and for acoustic phenomena that occur in the rela- tively long airways of humans. No models have yet been shown to be satisfactory for clearly discriminating between mechanical properties of central and peripheral air- ways of human lungs. It is not clear whether oscillation meas- urements will constitute improved tools for detecting and describing abnormali- ties of respiratory system mechanics due to environmental exposures. Considerable work remains to be done to develop appro- priate models and to confirm associations among impedance changes, physiologic correlates, and alterations in respira- tory system structure. That will require both application of the method to patients with known abnormalities of representa- tive types and the study of animals with specific, experimentally induced abnor- malities. Those lines of research are just now being pursued, and the utility of the approach is not likely to be fully known for a few years. Measurements of oscillation mechanics with substantially improved descriptive value beyond that of tests currently in use would require specialized equipment. The oscillating system would consist of computer-generated signals fed to care- fully calibrated loudspeakers or pumps. The frequency-response characteristics of the measurement system would have to be optimized. The data-reduction and mod- el-fitting systems would be computer- based. Although the equipment would be specialized, it could probably be packaged into a mobile unit that could be operated by people with only modest training. Pro- fessional input would be required for cali 49 bration, supervision of maintenance, and interpretation of results. In summary, oscillation mechanics has potential for development into a useful marker of response. Its advantage lies in its ability to distinguish mechanical abnormalities on a site-specific anatomic basis. Its primary disadvantages are its dependence on an appropriate model for fitting data and the likelihood of substan- tial variation among individuals in re- gional mechanical properties of the res- piratory system. General acceptance and widespread use will require substantial effort to demonstrate physiologic and clinical correlates, standardization of procedures and analysis, and packaging into measurement systems that are readily used. Static-Quasistatic Lung Pressure-Volume Analysis Lung compliance measured during breath- ing is usually lower than the actual com- pliance of lung tissue, because of the lack of time for tissue relaxation and because of differences in compliance among lung units. Measurement of static or quasi- static compliance avoids such frequency dependence by plotting transpulmonary pressure against lung volume during a single, slow exhalation. The current tests are assays that examine the elastic properties of lung tissue most directly and are the procedures of choice, if a spe- cific index of lung elastic recoil is de- sired as a marker of response. Standardized procedures for measuring lung compliance were recommended in a re- port from NIH (Macklem, 1974~. Transpul- monary pressure is measured with an esopha- geal balloon catheter (Dawson, 1982~. The subject inhales to total lung capacity and then exhales slowly while the exhaled volume and transpulmonary pressure are recorded. The test is termed quasistatic if exhalation is continuous, and static if exhalation is interrupted periodically to allow flow to cease and elastic forces to come to equilibrium. The elastic char- acteristics of the lung are expressed either by calculating compliance as the

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so slope of some portion of the pressure- volume curve or by simply displaying the entire curve. The lung pressure-volume curve shifts to the left (compliance increases) when lung elastic recoil is reduced (e.~.. in emphysema) and shifts to the right (compli- ance decreases) when elastic recoil is increased (e.g., in fibrosis) (Macklem and Becklake, 1963~. The curve represents the integrated elastic characteristics of the entire lung. It is not specific for the anatomic site of the change in recoil. Nor is it specific for the cause of the change in recoil. For example, fibrosis, inflammation, edema, and proliferative disorders could all cause similar shifts of the curve to the right (showing reduced compliance). Regardless, the test could be a useful marker of response in popula- tions in which abnormal elastic recoil is a likely response. Intrapulmonary Gas and Particle Distribution Gas Distribution Properties- Single-Breath Gas Washout Patterns of the washout of gases inhaled in a single breath have received consider- able attention as indexes of small airway disease. Although the test can be perform- ed by having the subject inhale a bolus of inert gas (bolus technique), the most com- mon approach is to evaluate the washout of nitrogen from the lung after an inhala- tion of oxygen (resident-gas technique). The single-breath nitrogen washout (SBNW) test was introduced in 1969 as a test of small-airway function (Anthonisen et al., 1969~. The air-breathing subject exhales to residual volume, inhales a sing- le breath of oxygen, and exhales again to residual volume. The nitrogen concentra- tion of the expirate is plotted against its volume. The normal curve has a charac- teristic shape, first noted in 1949 (Fowl- er, 1949), in which the nitrogen is ini- tially low (washout of dead-space oxygen), rises to a plateau that represents the nitrogen-oxygen distribution in the ma- jority of the lung, and then increases again near the end of the exhalation. The M4RKERS IN PULMONARY TOMCOLOGY slope of the curve depends on the uniform- ity of gas distribution among ventilating units and is affected both by asymmetry of airway path lengths and by nonuniformity of compliance among ventilating units (Engel and Macklem, 1977~. The slope in- creases as gas distribution becomes less uniform. The onset of the terminal nitro- gen rise has been termed "closing volume" and is thought to indicate the lung volume at which airway closure begins (Engel et al., 1975~. The phenomena responsible for determining the closing volume remain incompletely defined, but it is generally agreed that an increase in, if not onset of, airway closure is primarily respon- sible (Forkert et al., 1979~. It is interesting that Ernst et al. (1986) examined the relationship of clos- ing volume and fluoride air pollution in children living near an aluminum smelter. In both sexes, there was a significant linear relationship between increased closing volume and the amount of fluoride found in urine samples from the children. Since 1969, the SBNW test has been the focus of numerous physiologic studies and has been applied in several population studies. Standardized measurement proce- dures were disseminated by NIH in 1973 (Martin and Macklem, 1973), partly to fa- cilitate multi-institutional collabora- tive studies funded by the National Heart and Lung Institute (NHLI). Methods for computerizing analysis of the curves have been published (Craven et al., 1976; Cramer and Miller, 1977~. Equipment for perform- ing the test is available commercially, and several equations have been developed for predicting normal values of SBNW para- meters (Gold, 1982~. Although the SBNW test continues to be included in lists of tests sensitive to small-airway disease, its usefulness as a marker of responses to environmental exposures has not been clearly demonstrat- ed. In 1973, a workshop on screening pro- grams for early diagnosis of airway ob- struction (NHLI, 1973) concluded that, "although closing volume and closing capa- city are sensitive tests, they are probably of low specificity and moderate precision, and their validity as an early diagnostic test is unknown." The presumed usefulness

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PHYSIOLOGYININTACT ORGANISMS of the test is founded largely on the find- ing in numerous studies that it can detect abnormalities in asymptomatic smokers, often in the absence of abnormalities in "conventional" lung function tests (McCarthy et al., 1972; Buist and Ross, 1973; Nemery et al., 1981; Teculescu et al., 1986). Recent work has more directly demon- strated associations between SBNW abnor- malities and small-airway pathologic conditions. Cosio et al. (1978) and Berend et al. ~ 1981 a,b) found significant cor- relations between abnormal values of wash- out slope and closing volume in human sub- jects and small-airway disease in excised lung tissue. The latter study demonstrated that closing volume was related more close- ly to small-airway inflammation than to lung elastic recoil. Petty et al. (1980) performed SBNW tests on excised human lungs and found that increased closing volume was associated with inflammation and squa- mous metaplasia in small airways. Those results further confirm and define the morphologic basis for SBNW abnormalities. Incalzi et al. (1985) recently published regression equations of SBNW parameters with age, height, and lung volume for 234 normal subjects 20-80 years old with no history of smoking, occupational exposure to known pulmonary toxicants, or chronic respiratory illness. The authors conclud- ed that the variability was too great for detection of subtle changes in population studies. In summary, the utility of the SBNW test as a marker of responses to environmental exposures remains uncertain. The test reflects small-airway abnormalities, but its sensitivity and specificity are questionable. Inhaled-Particle Distribution The deposition of inhaled particles is a function of particle characteristics, airway geometry, and ventilation. The latter two can be altered by exposure to pollutants or by disease, so it follows that aerosols can be used as markers of exposure or response to environmental agents. Although the specific equipment required for tests of aerosol distribution 51 is not generally available in the standard pulmonary function setting, the technol- ogy is neither new nor very complicated. Establishment of guidelines for their use, which do not now exist, could result in uniform application in the future. Aerosol particles can be used to assess pulmonary structure and function, because they can trace the convective motion of air in the lungs and their deposition is related to the dimensions of the airways through which they pass. Three techniques can be used to obtain information from inhaled particles; they allow assessment of airway sizes and inhomogeneities of ventilation and gas mixing. The use of aerosols to assess mechanical clearance from the respiratory tract is discussed later. Assessmentof gasmixing. Intrapulmonary mechanical mixing of gases can be assessed by injecting an aerosol during the entire tidal-volume inhalation or in a pulse dur- ing a portion of this inhalation and then examining the particle concentration recovered in exhaled air. The procedure requires the use of particles with a mini- mal probability of deposition-approxi- mately 0.3-0.5 ,um-so that their loss from the inhaled air occurs largely because of nondiffusive gas mixing in the lungs, i.e., bulk transfer from tidal to reserve air. By separating mixing due to molecular diffusion from mechanical mixing due to airflow, one can estimate the role of molecular diffusion in ventilation (Alt- shuler et al., 1959~. Although the proced- ure provides some assessment of bulk transfer, conclusions as to the sites at which this occurs await the further devel- opment and use of models of aerosol dynam- ics and gas transport in the lungs that take into account the effects of geometric com- plexities on lung ventilation (e.g., Engel,1983~. The distribution of exhaled aerosol in normal people shows a fair degree of inter- subject variability; nevertheless, gener- al profiles are reproducible within groups of subjects, and exhaled-aerosol measure- ments have been used to assess airway ab- normalities, in which case the shape of the aerosol exhalation curve is different from that in normal subjects. In airflow

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52 obstruction, for example, the shape of the curve is different because the recovery of particles is decreased, owing to an increased rate of particle deposition and a change in mixing characteristics. A correlation has been found between the percentage of aerosol recovered and the predicted percentage change in FEVER over a wide range of degrees of airway obstruc- tion (Muir, 1970~. A difference in aerosol recovery (i.e., a decrease) has also been demonstrated in coal miners with various forms of pneumoconiosis (Hankinson et al., 1979~. Aerosol probe procedures. The aerosol probe technique allows inferences con- cerning small-airway (< 1 mm) and al- veolar dimensions; it is of particular use for assessing changes in diameter, such as those associated with obstructive lung disease. Conducting-airway obstruc- tion can also be detected with conventional pulmonary function tests, but the latter might be less sensitive than the probe procedures in detecting early changes. However, alveolar size, which is important - in assessing emp ~ysema progression, can be estimated in viva only with particle probes. The aerosol probe procedure is a modifi . ~. ~ cation of the aerosol mixing technique discussed above, in that a period of breath-holding is generally imposed be- tween aerosol inhalation and exhalation. It is based on the concept that the amount of deposition of inert, nonhydroscopic monodisperse particles with a known, but low, rate of sedimentation during the breath-holding period depends on the set- tling distance required for the particles before they come into contact with an air- way wall; this distance is a reflection of the overall dimensions of the airspaces in which the aerosol is found. Particles that are deposited and are removed from the air will not be recovered during ex- halation. In reality, and as should be evident from the discussion of the mixing technique above, not all the inhaled aero- sol will be recovered even if there is no breath-holding period between inhalation and exhalation. In practice, the aerosol probe procedure requires determination of the extra loss of particles due to gravi AL9RKERS IN PULMONARY TOXICOLOGY rational settlement. The ideal particle size range used in the procedure is 1-1.5 Am MMAD (Gebhart et al., 1981~; however, the increased impaction deposition in people with obstructed airways might re - Cure some size adjustment. The work of Palmes et al. (1967) estab- lished a basic method for estimating the effective dimensions of the respiratory airspaces with monodisperse aerosols. An aerosol is inhaled; the breath is then held at close to total lung capacity (TLC), with different breaths held for periods of 0-30 seconds; and a volume equal to twice the inhaled volume is then exhaled, to ensure recovery of all remaining airborne particles. The persistence of the aerosol-i.e., the probability of its remaining suspended in air and thus being exhaled-decreases exponentially as a function of breath-holding time. Aerosol inhalation was completed near TLC, so most of the aerosol mass and most of its deposi- tion were assumed to be in the region of the respiratory bronchioles and alveolar ducts; the contribution of anatomic dead- space volume was considered to be small, and its influence on the shape of the aero- sol recovery curve could be ignored. The logarithm of percent of aerosol recovery is plotted against breath-holding time; this results in a curve whose slope (or slopes) is related to the average size of the airways within which residual aerosol remained before exhalation. Results are usually expressed in terms of the half- time of aerosol persistence in the lung. Subjects differ substantially, but the aerosol probe procedure is sensitive to changes in airway dimensions and does yield reproducible results in normal subjects repeatedly tested (Lapp et al., 1975~; in addition, the variability in airway size measured in healthy people was found to be quite similar to that measured in fixed lungs obtained from accident vic- tims. However, the wide intersubject vari- ability in normal people is a negative feature of the test if applied to people with lung abnormalities; e.g., in obstruc- tive disease, the results can be equivocal (Palmes et al., 1971, 1973~. In some cases, increased half-time of aerosol persis- tence indicates enlarged airspaces; but

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PHYSIOLOGYIN INTACT ORGANISMS results in patients with diagnosed emphy- sema can be comparable with those in healthy people. Inasmuch as the aerosol has access only to airspaces to which it is delivered by convective airflow, it can penetrate to either predominantly normal or diseased lung tissue, depending on the site of airflow obstruction (if any). Thus, half-times observed in pa- tients are more variable than those observ- ed in healthy subjects. In addition, the amount of aerosol at zero time of breath- holding was generally lower in patients than in normal persons; that indicates increased deposition during the dynamic phase of the breath-holding maneuver, probably due to obstruction and the result- ing narrowing of airways. Another group examined with the aerosol probe were coal miners with pneumoconiosis (Hankinson et al., 1979~. There was some correlation between disease type, aerosol persistence, and calculated airway dimen- sions, but a lack of correlation between persistence and recovery suggested either that the mechanisms that cause changes in those two phenomena are differ- ent or that the changes occur at different sites in the respiratory tract. The latter possibility is of concern, because the results of the tests are a reflection of airway dimensions at various depths In the lungs. A slight modification of the aerosol probe technique might be used to examine particular regions (depths) in the lungs. The modification, known as the bolus probe technique, involves either inhalation of an aerosol bolus followed by a preset (but variable) volume of particle-free air (Palmes et al., 1973) or inhalation of the bolus at various stages of inhala- tion rather than at a particular fixed stage (Heyder, 1983~. When this procedure is used, inhalation of equivalent tidal volumes leads to decreases in measured effective airway diameter as lung volumes decrease. In addition, the measured per- sistence and thus the actual average airway diameter that is measured depend heavily on the inhalation volume containing the aerosol, in that, the more deeply a bolus is inhaled, the greater is the dispersion of the bolus later exhaled and the smaller 53 are the airways being "probed." The airway dimensions calculated from aerosol recov- ery curves for different depths of inhala- tion have been found to agree well with what would be expected, on the basis of comparisons with both morphometric models of the human lung and measured airways in fixed lungs (Gebhart et al., 1981; Heyder 1983; Nikiforov and Schlesinger, 1985~. In an aerosol rebreathing procedure described by Kim et al. (1983), subjects breathe 1-,um particles 30 times/minute from a volume held at 500 ml. That results in magnification of the differences in particle recovery after a single breath. The test is able to screen reproducibly for airway constriction and might also indicate the extent of such concentration; variability in both normal subjects and those with chronic obstructive airway disease was 5-10%. The ability of the test to detect changes early in pathogenesis is not yet known (C. S. Kim et al., 1985~. A variation of the single-breath aerosol bolus technique that is sensitive in de- tecting early airway changes has been de- scribed (McCawley and Lippmann, 1984; McCawley, 1987~. It involves precise con- trol of volume and flow during introduction of a bolus of monodisperse (0.5-pm MMAD) particles. With a dispersion index, it was possible to differentiate between healthy smokers and nonsmokers; thus, the procedure seems capable of detecting the early changes known to occur in the small airways of smokers. There were found to be no differences in tests of forced ex- piration (e.g., FIVE andFVC) betweensmok- ers and nonsmokers, and the coefficient of variation of the dispersion parameter (approximately 17% in nonsmokers and 36% in smokers) was less than that of the pul- monary mechanics tests. The procedure optimizes the protocol for rapid screening and is useful in epidemiologic studies that attempt to assess airway obstruction. Regardless of the specific probe proced- ure used, there are always differences between the simple theoretical assump- tions of the models of aerosol deposition and actual complex situations. Direct anatomic interpretations should be made with caution, and one must bear in mind effects of a number of factors, e.g., axial

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72 MARKERS IN PULMONARY TOXICOLOGY Zo~ Z ~ IS ~ I,V. Oleic Acid (4) ~ ~ '~~~ ~ ~ PEEP (8) 6 Z ~ PEEP (8) L ~ 20 ~ I k1 = 7 9 + 2.9 %/min \\ 50 I 1 1 1 10 ~I I 0 1 2 0 1 2 HOURS FIGI~E 3-4 Albumin clearance discnm~nates between lung injury and lung inflation. such as epinephrine, changes in endotheli- um and changes in other cell types seem to occur simultaneously. The location of the earliest damage to pulmonary endothelium might also very from one toxicant to another. For example, in oxygen toxicity, capillary endothelium is the first to show changes; after bleomy- cin administration, the earliest damage is to arterial and venous endothelium. Thus, markers that could distinguish in- jury in various parts of the vascular bed might provide important clues to mechan- isms and risks associated with various pulmonary diseases. Loss of Endothelial Barrier Function A major role of endothelium is to prevent loss of fluid from vessel lumina. Endo- thelial cell injury is usually manifested clinically as evidence of pulmonary edema. Pulmonary edema is classified into two types. In hydrostatic or hemodynamic edema, abnormally high intravascular pressures in small parenchymal vessels lead to flux of fluid from them. In permea HOURS FIGURE ~5 Compartment analysis of DTPA clearance. bility edema, intravascular pressures can be normal, but leaks in alveolar capil- laries allow increased flux of water and protein into the extravascular compart- ment. The former type does not always en- tail injury to the vascular endothelial barrier, but can arise, for example, as a result of constriction of pulmonary ven- ules. The most common examples of hemody- namic pulmonary edema result from chronic left-sided heart failure or mitral valve disease (Fishman, 1980~. Permeability edema is associated with formation of a protein-rich lymph that arises from an injured endothelial barrier that allows increased passage of plasma proteins into the interstitium of the pulmonary paren- chyma. Permeability edema results from exposure to some noxious airborne agents, such as nitrogen dioxide, and also occurs in acute respiratory diseases. In animal studies, it also results from a number of chemical insults to pulmonary capillary endothelium, e.g., after exposure of the pulmonary vasculature to oleic acid, al- loxan, a-naphthylthiourea, or phorbol ester.

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PHYSIOLOGYININTACT ORGANISMS In animals, permeability edema has been measured on the basis of protein leakage from the pulmonary vasculature. Lung lymph flow, lymph fluid protein concentration, and accumulation in excised lungs of radio- labeled protein introduced into the blood all can be measured. They have provided much important information on vascular leak in laboratory animal studies, but not directly in humans. Loss of barrier functions is also re- flected in an increase in extravascular lung water. Regardless of the cause, ex- cess extravascular water can be detected with a variety of methods. In general terms, those methods can be divided into invasive and destructive methods, inva- sive and nondestructive methods, and non- invasive and nondestructive methods (Table 3-2). The destructive techniques 73 dyspnea and tachypnea) and physical find- ings of cyanosis and rates during chest auscultation (Staub, 1974, 1986~. How- ever, other conditions can cause similar findings. In any case, the approach is nonquantitative and relatively insensi- tive to smaller accumulations of extravas- cular water. Nonetheless, because of its simplicity and lack of expense, the clinical examination remains an Important means for detecting the presence of acute pulmonary edema. Of the other available techniques, four deserve special consideration: chest roentgenography, the indicator-dilution method, and the newer techniques of posit ron-emission tomography and nuclear mag netic resonance. Chest roentgenography has many favor able features for use as a marker of lung have the mayor advantage ot accuracy and injury. It is practical, widely available thus are often reported as the putative in a variety of useful settings, and rela "gold standard" by which other techniques lively inexpensive. Its accuracy and sen are judged (Staub, 1974; 1986~. Clearly, sitivity in detecting pulmonary edema are disputed. When strict attention is paid to technical factors, some have argued that the chest roentgenogram is quite sen sitive to changes in lung water content, and accurate inferences can be made about the magnitude of such changes (Pistolesi and Guintini, 1978; Milne et al., 1985). However, several other groups have tested it against presumably more accurate tech however, they are unsuitable for clinical studies. The nondestructive techniques, although useful in a clinical setting, suffer to various degrees from inaccuracy, non- specificity, impracticality, and expense. For instance, pulmonary edema can be diag- nosed clinically in a patient with a char- acteristic history (e.g., acute onset of TABLE 3-2 Methods for Detecting Excess Extravascular Lung Water Accumulation Category Methods Invasive and destructive Invasive and nondestructive Noninvasive and nondestructive Gravimetrics (Staub, 1974,1986) Histology (Staub, 1974, 1986) Indicator dilution (Baudendistel et al., 1982; Grover et al., 1983; Sibbald et al. 1983; Eisenberget al., 1987;Sivak and Wiedemann, 1986;Effros, 1985;Lewis et al., 1982) Clinical examination (Staub, 1974, 1986) Pulmonary mechanics (Staub, 1974, 1986) Chest roentgeno~aphy Mane et aL, 1985; Pistolesi and Guintini, 1978; Baudendistel et al., 1982; Grover et al., 1983; Seybold et al., 1983; Eisenberg et al., 1987; Sivak and Wiedemann, 1986) Soluble-gas uptake (Overland et al., 1981) Microwave transmission (Iskander et al., 1979) Compton scatter (Loo et al., 1986) X-ray computed tomography (Hedlund et al., 1984; 1985) Positron emissiontomography(Schusteretal., 1985;Rhodeset al., 1981;Wollmer et al., 1984; Schober et al., 1983; Schuster et al., 1986; Cutillo et al., 1984) Nuclear magnetic resonance (Cutillo et al., 1984; Morris et al., 1985; Wexter et al., 1985)

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74 MARKERS IN PULMONARY TOXICOLOGY niques and have not been able to demon- called the thermal-green dye double-in strate an acceptable degree of accuracy dicator dilution technique, has been veri (Baudendistel et al., 1982; Grover et al., fled by numerous groups as accurate (in 1983; Sibbald et al., 1983; Sivak and most. although not all. instances in which Wiedemann, 1986; Eisenberg et al., 1987~. Two techniques, indicator dilution and positron-emission tomography, measure the intravascular components of lung water. Thus, they can measure extravas cular lung water accumulation, which is in fact the entity of interest, inasmuch as the abnormal accumulation of extravas cular water represents breakdown in en dothelial cell barrier function. In addi tion, gas rebreathing techniques have been used to estimate lung tissue volume and pulmonary capillary blood volume. From those two volumes and estimates of ratios of wet to dry weight of tissue and blood, intravascular and extravascular water can be estimated. Indicator-dilution methods of measuring extravascular lung water are based on the concept that the mean transit time of an indicator through a fluid depends on in dicator flow rate and the volume of the fluid (Lewis et al., 1982; Hedlund et al., 1984; Sivak and Wiedemann, 1986~. For a given flow rate, if volume is small, the mean transit time will be small, and vice versa. To measure extravascular lung water, two indicators are used: one that can diffuse through the entire lung water volume and one that is limited to the in travascular, nondiffusible volume. A1 though a number of indicators have been used, the two that have achieved the great est acceptance are heat (actually, temper ature change) as the diffusible indicator and dye (e.g., indocyanine green) as the nondiffusible indicator. The green dye binds immediately in viva to albumin and thus remains intravascular during the period of lung water measurement. Extra vascular lung water (EVLW) can be calcu lated as EVLW = CO (MTT~ - MTTg&), where CO is the cardiac output (i.e., a measure of vascular flow) and MTT is the mean transit time of the thermal (t) or green dye (ad) indicator. This method, EVLW is increased), reproducible, and reasonably sensitive to changes in EVLW (i.e. it will reliably detect approximate- ly a 20% change) (Sivak and Wiedemann, 1986~. Nonetheless, it is moderately in- vasive (catheters in the pulmonary and femoral arteries are required) and thus is not suitable for general population screening studies. Positron-emission tomography (PET) is a nuclear-medicine technique that pro- duces quantitative tomographic images of the tissue distribution of a previously administered positron - emitting radio - nuclide. It uses image reconstruction algorithms identical with those used dur- ing routine x-ray computed tomography (CT). However, unlike x-ray CT, which cannot distinguish between intravascular and extravascular water (Hedlund et al., 1984, 1985), PET measurement of EVLW is feasible because it subtracts the intra- vascular water content (IVW) of a region from the total lung water content (TLW) of the same region (Schuster et al., 1985~. Unlike the indicator-dilution method previously described, PET is less sensi- tive to errors caused by the underestima- tion of EVLW in poorly perfused areas of lung. The intravascular component of EVLW is measured during PET by scanning the subject at least 2 min after inhalation of i50- labeled carbon monoxide, a gas that avidly binds to hemoglobin. IVW is calculated by comparing the radioactivity in a given lung region with activity in blood samples taken during the scan. A similar procedure is used to measure TLW, except that the scan is obtained during equilibrium of the bolus infusion of )50-labeled water. The cal- culation of extravascular water content of a region involves the subtraction of IVW from TLW. Alternatively, a constant infusion of )50-labeled water, or iiC- in- stead of )50-labeled carbon monoxide or density measurements instead of TLW meas- urements may be used (Rhodes et al., 1981; Schober et al., 1983; Wollmer et al.,

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PHYSIOLOGYIN INTACT ORGANISMS 1984~. Recent studies in whole animals have suggested that PET provides measure- ments of EVLW in both normal and edematous lungs with acceptable accuracy and is sen- sitive to small changes in EVLW after phys- iologic intervention (Schober et al., 1983, Schuster et al., 1986~. Values ob- tained in humans have been comparable with those obtained in experimental animals. PET appears to be ideal for measuring regionalEVLW content. Because of technical problems associated with radioactivity counting in heterogenous tissues, whole- lung values for EVLW are more difficult to obtain. More important, however, are the cost and impracticality of PET as a clinical tool in that a scanner, a com- puter, a cyclotron, and several highly trained personnel are required for obtain- ing the measurements. Proton nuclear magnetic resonance (NMR) imaging is a new, complex, and expensive technique for evaluating lung water con- tent (Cutillo et al., 1984; Wexter et al., 1985~. NMR depends on the electromagnetic properties of nuclei of some atoms that cause them to act like small, spinning bar magnets when placed in a strong magnetic field. The most abundant of those atoms is hydrogen, which contains one proton. The proton is the principal nucleus used in current magnetic resonance Imaging experiments. When it is placed in a strong magnetic field, there is a slight net or- ientation of the protons along the magnetic field direction. The introduction of a ,= radiofrequency (RF) excitation at a fre- quency specific for both the magnetic field strength and the protons under considera- tion causes the reorientation of the pro- tons; when the RF excitation is removed, the protons return to their original orien- tat~on. ~ hat process (i.e., return, or relaxation, of the proton) emits RF energy, which is detected by a sensitive antenna or coil, amplified, and processed by a computer. The computer processing of space- and time-dependent RF emission creates an image of the concentration (i.e., density) and environment of protons in fat and water of soft tissue. Desirable features of proton NMR imaging are that no ionizing radiation is necessary and 75 there are no bone artifacts in the image. Although Now imaging will probably yield the most accurate in viva measurement of lung-water distribution, subtraction of the vascular component remains diffi- cult. That problem, signal-to-noise ratio characteristics in the imaging of lung tissue, and the complexity of the technol- ogy as a whole make NMR imaging, like PET imaging, unlikely candidates for screen- ing general populations for evidence of endothelial lung injury. It is probably unwise to use lung-water measurements obtained with any technique to evaluate early lung injury. The abnor- mal accumulation of excess lung water rep- resents not only a failure of endothelial barrier function, but also a failure of various other mechanisms (the most impor- tant of which is lymphatic function) that the lung can use to maintain normal water homeostasis. More useful as a marker of early injury would be a technique that detected breakdown of endothelial barrier function itself. Several groups have measured the flux of radiolabeled proteins across the pulmonary endothelium with external radiation detectors of various sorts (Gorin et al., 1978; Mintun et al., 1987~. Although those techniques indeed seem to be more sensitive markers of lung injury than is the measurement of EVLW, they are still too new for prediction of how accurate, reproducible, and practical they will be in detecting lung injury in groups of humans. In summary, no ideal means exists, or is likely to exist in the near future, for the detection of lung endothelial injury on the basis of either lung-water or capil- lary protein-flux measurements. The tech- niques that are simple, inexpensive, and practical to apply to large groups of hu- mans are generally nonspecific and insen- sitive. The techniques that improve on specificity and sensitivity suffer in being expensive, impractical, and com- plex. The choice of method will depend largely on the specific goals of the pro- gram involved.

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76 Nonbarrier Properties of Endothelium As noted above, the nonspecificity, insensitivity, invasiveness, require- ments for sophisticated equipment, and expense of currently available measures of endothelial barrier function limit their usefulness in diagnosing early per- meability defects or subtle endothelial cell injury that can be associated in some people with a predisposition to serious pulmonary vascular disease. Obviously, markers associated with subtle, early defects in pulmonary endothelium that are sensitive, specific, and minimally in- vasive could be useful in identifying people at risk. Similarly, predisposition of people to diseases associated with de- fects in the endothelium might be predicted and such diseases prevented more effec- tively. For example, diverse types of trauma result in adult respiratory dis- tress syndrome (ARDS) in some patients. Markers to identify subtle changes in en- dothelium might aid in identifying pa- tients at risk of developing ARDS and in understanding its pathogenesis. Research during the last several years has led to the identification of several non-barrier functions of pulmonary en- dothelium. From the standpoint of increas- ing our knowledge of mechanisms of lung injury, there is a need to understand bet- ter both barrier and nonbarrier functions of endothelium, to attain the capacity to assess them, and to determine how non- barrier functions of endothelium are cor- related with barrier properties. It should be recognized that changes in nonbarrier functions of endothelial cells might be useful predictors of deficits in the bar- rier function of the endothelium. Metabolic Activity of Endothelium The pulmonary vasculature performs a number of potentially important nonbar- rier functions, some of which involve the modification of circulating concentra- tions of naturally occurring, biological- ly active substances, as well as drugs. Because the lung has a large vascular sur- face area and receives all of the cardiac MARKERS IN PULMONARY TOXICOLOGY output, it is uniquely situated to alter rapidly the circulating concentrations of vasoactive agents before they reach the arterial circulation. The capacity of the lung to clear the circulation of chemical agents and the potential impor- tance of this function have been the sub- ject of several reviews (Gillis and Pitt, 1982; Roth, 1985). The ability to remove and metabolize substances reflects properties of endo- thelial cells of small vessels and capil- laries in lung. For example, carrier- mediated transport of biogenic amines, such as 5-hydroxytryptamine (SHT) and norepinephrine (NE), into pulmonary vascular endothelium occurs. Available evidence indicates that SHT and NE are taken up at different sites at the endothe- lial surface. After removal by the lung vasculature, those amines are metabolized by enzymes like monoamine oxidase and catechol-O-methyltransferase. However, the rate-limiting step in their initial removal from the circulation is transport from the vascular space, rather than intrapulmonary metabolism. Circulating adenine nucleotides (adeno- sine monophosphate, adenosine diphos- phate, adenosine triphosphate) are also altered on passage through the lung. Aden- osine triphosphate, for example, does not survive passage through the pulmonary circulation. Biochemical and cytochemi- cal studies have shown that, when those nucleotides are perfused through isolated lungs, all the radioactivity entering the pulmonary circulation is recovered in the effluent, but none remains in the form of the adenine nucleotide. The mean transit time and volume of distribution of those nucleotides are the same as those of in- travascular markers. This indicates that the adenine nucleotides are metabolized in the pulmonary circulation without leav- ing the vascular space. Cytochemical data confirm that, although several cell types and organelles have phosphate esterases that hydrolyze nucleotides, only the en- zymes that face the vascular lumen are exposed to and metabolize them. The loca- tion of the enzymes along the vascular lumen accounts for the fact that the meta

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PHYSIOLOClYININTACT ORGANISMS boric products of adenine nucleotides appear in the venous circulation with no delay or tissue uptake. The lung is also capable of hydrolyzing circulating peptide hormones, such as bradykin in and angiote ns in I. B radykinin is nearly quantitatively converted to shorter peptides in a single pass through the pulmonary circulation. The peptide is not taken up by lung, and its mean transit time and volume of distribution in perfused lung preparations are identical with those of intravascular markers, such as indocya- nine green or blue dextran. Similarly, angiotensin I is extensively converted to angiotensin II on passage through the pulmonary circulation. Angiotensin- converting enzyme is located on the luminal surface of pulmonary endothelium; indeed, immunohistochemical studies have confirm- ed pulmonary endothelium as the only site of angiotensin-converting enzyme in the lung. Studies in animals have revealed that impaired pulmonary metabolic function results from exposure to numerous toxi- cants. However, structural injury to pul- monary endothelium is not always asso- ciated with deficits in each type of metabolic function. For example, the pyrolizidine alkaloid, monocrotaline, produces pulmonary endothelial injury experimentally that is associated with reduced intrapulmonary clearance of SHT by isolated lungs from treated animals (Roth, 1985~. However, 5'-nucleotidase and angiotensin-converting enzyme ac- tivities in isolated lung preparations are apparently unaffected by treatment of rats with monocrotaline. Thus, chemi- cally induced damage to lung might affect some functions of endothelium without altering others. This suggests some specificity in the endothelium-damaging action of some toxicants. A number of studies have suggested that pulmonary metabolic functions may provide sensitive markers of endothelial injury. For example, exposure to the herbicide paraquat results in pulmonary lesions in humans and experimental animals. In rats, marked structural changes in alveolar epithelium have been commonly observed 77 after paraquat administration, but al- terations in vascular endothelium are much more subtle and infrequent. A modest but reproducible decrease in the ability of isolated lungs from paraquat-treated animals to remove perfused 5HT has been reported (Roth, 1985~. The demonstration of impairment in SHT clearance resulting from a treatment that produces little, if any, structural alteration in endothel- ium suggests that the pulmonary metabolic function could be a sensitive index of damage to pulmonary endothelium under some circumstances. That view is supported by studies of oxygen toxicity. Structural alterations in pulmonary capillary en- dothelial cells are an early manifestation of exposure to oxygen at 1 atmosphere. Block and Fisher ( 1977) reported that, although ultrastructurally demonstrable endothelial damage is not apparent until 48 hours of exposure to 100% oxygen, ex- posure for as little as 18 hours produces a significant decrease in SHT clearance by lungs of exposed animals. Those studies of pulmonary metabolic function in animals were performed in iso- lated lung preparations. The functions have also been studied in vivo both in ani- mals and in humans with the multiple-in- dicator-dilution techniques described previously. For example, angiotensin- converting enzyme activity in the pulmon- ary vasculature has been studied with the synthetic substrate 3H-benzoyl-phe-ala- pro (BPAP). BPAP and an intravascular marker are injected intravenously as a bolus, and the concentrations in the arter- ial (i.e., postlung) blood are compared over time. With this technique, the frac- tion of BPAP metabolized in a single pas- sage through the pulmonary vasculature can be calculated. There are potential pitfalls in using that and related methods to assess pulmon- ary microvascular injury (Stalcup et al., 1982~. For example, pulmonary metabolic function can be influenced by changes in transit time and by inhomogeneity of perfusion, edema, and other factors that affect vascular surface area. When exogen- ously administered, radiolabeled sub- strates (e.g., 3H-BPAP) are used, it is

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78 possible for endogenous substrates (e.g., angiotensin I and bradykinin) to compete with the tracer for metabolism and thereby confound interpretation of results. Fur- thermore, the lung might simultaneously synthesize and release the same test sub- stance being removed or metabolized, and that would make interpretation of pulmon- ary extraction data difficult. In addi- tion, questions have been raised about how to normalize metabolism data (e.g., whole lung vs. per unit lung weight, pro- tein, DNA, etc.~. Careful monitoring of perfusion, in- travascular pressures, and ventilation aid somewhat in ensuring reliability of data but do not resolve many of the poten- tial problems. As mentioned above, the choice of a specific metabolic function and substrate can be of critical importance with regard to usefulness of a metabolic process in assessing lung microvascular function. For example, substrates that are removed entirely in a single pass through the pulmonary vasculature might not provide needed sensitivity. In this case, reductions in enzymatic capacity might have to be quite large for effects on intrapulmonary metabolism to be detectable. Some of the potential pitfalls can be addressed through refinements in tech- niques. Indeed, if BPAP doses that provide both saturating and nonsaturating con- centrations of substrate at enzyme sites in the pulmonary vasculature are measured serially, enzyme kinetics can be deter- mined from the resulting indicator-dilu- tion curves. Thus, the Michaelis constant (Km) for the enzyme can be calculated, as can Amp which is the product of the maxi- mal velocity (Vm:,x) of the reaction and the microvascular plasma volume. From those estimates, information can be ob- tained on changes in enzyme quality (as measured by affinity) and amount (as meas- ured by Vmax) in toxicoses or other dis- ease states. With angiotensin-converting enzyme (ACE), for example, a reduction in Ama,, could reflect specific inhibition or destruction of the enzyme or a decrease in capillary surface area. Changes in Km, however, reflect alterations in endothel M'4R=RS IN PULMONARY TOXICOLOGY . ial metabolic function that are indepen- dent of effects on capillary surface area. This technique has been used to study effects of pneumotoxicants and ACE inhibi- tors on pulmonary endothelium. Indeed, alterations in metabolic function of en- dothelium have been described for such toxicants as PMA and nitrofurantoin and for radiation-induced injury. Studies in rabbits, for example, revealed an in- crease in Km for BPAP soon after administra- tion of PMA when no histologic evidence of lung injury was observed (McCormick and Catravas, 1986~. The data suggest that, under some circumstances, pulmonary metabolic function can provide a sensitive index of injury to pulmonary endothelium. As with several other potential markers of lung injury, the use of pulmonary meta- bolic function to assess endothelial injury in the lung requires further devel- opment and validation before it can be con- sidered useful. The equipment and techni- cal sophistication required to perform such assessments are considerable, so modifications would clearly be needed if the method were to be used in routine clini- cal or screening situations. Thus, it is clear that measurements of pulmonary meta- bolic functions or other nonbarrier func- tions of endothelium have not reached the status of clinically useful, diagnostic tests. However, with recent and forthcom- ing advances in technology, it is not out- side the realm of possibility that such techniques will be useful both in the clin- ic and in the field. Some of the needs for future research and development are increased basic knowl- edge of how nonbarrier endothelial func- tions, such as transporters and enzymes, work in vivo; investigation in animal mod- els of how acute and chronic lung injury changes several endothelial metabolic functions, especially in the absence of surface area phenomena; determination of the specificity and sensitivity of vari- ous probes in various injury models with the goal of matching the cause of injury with the probe; and simplification of tech- niques to make them more useful in human applications.

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PHYSIOLOGYININTACT ORGANISMS 79 TABLE 3-3 Summary of Charactenstics of Physiologic Assays Characteristicsa and Ratings A B C D E F Measure Respiratory function Spirometry Lung mechanics Dynamic compliance, resistance, + + and conductance Oscillation impedance Static pressure-volume Intrapulmonary distribution Single-breath gas washout + Particle distribution Exhaled particles Particle deposition Alveolar-capillary gas transfer CO diffusing capacity Exercise gas exchange Airway reactivity ++ + ++ + ++ + + + ++ ++ + + + ++ ++ + ++ + + + + + + + + + ++ + + + ++ O + O + + + + + + + ++ + ++ + + + + + Nonspecific reactivity + + +- + + + + + + Specific reactivity + + + +- + + + + Particle clearance Radiolabeled aerosol Magnetopneumography Air-blood ba'Tier function Conductingairway permeability Clearance of inhaled DTPA + Transepithelial potential + Alveolar permeability by + radiolabeled aerosol Vascular permeability Radiolabeled protein leakage Chest x ray for edema Extravascular lung water by indicator dilution, PET, or NMR + + + + + + + ++ ++ ++ + + + + + O + o + + O + + + + - Rebreathing soluble gases + + + +- + + + Endothelial metabolic function + + + - +- + aCharacteristics: A. Current State of Development. Considerations in this category included the number of groups using the tech- nique, the availability of the required equipment, the magnitude of the present data base, and the degree of standardiza- tion of procedures. B. Estimated Potential for Development. This category reflected the current estimate of the potential for substantial development of the assay beyond its present state. Although it was recognized that advancements are possible for any assay, this category was intended to reflect potential for substantial technical refinements, adaptation for use in large populations, or advancements in ability to interpret results. C. Current Applicability of Assay to Humans. Primary considerations were the invasiveness of the technique and the requirement for radionuclides. All the assays can be applied to animals, but some are less suitable than others for evaluating humans. D. Suitability for Measuring Large Numbers of Subjects. The focus of this category was the suitability of the assay for use in studies of large populations of people, as might be required for evaluating effects of some environmental expo- sures. Considerations included adaptability of equipment for mobile use, length and nature of subject interaction (i.e., degree of cooperation required), resources required to analyze samples and data, and subject safety. For example, a low rating might suggest a low suitability for field use in evaluating hundreds of subjects of various ages and both sexes, whereas the assay might be quite suitable for studies of dozens of selected subjects brought to a stationary facility.

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80 AilARKERS IN PULMONARY TOMCOLOGY E. Reproducibility. This category focuses on the variability of results within and between subjects. F. Interpretability. This category reflects the current understanding of (and degree of consensus as to) pathophysio- logic correlates, anatomic sites of effect, and causative agents. For many of the assays, there is little disagreement on the physiologic function affected, but the specific mechanism or site of change is uncertain. For example, it is agreed that reduced carbon monoxide diffusing capacity reflects reduced efficiency of alveolar-capillary gas transfer, but the test does not distinguish among the effects of a thickened membrane, reduced surface area, and reduced capillary blood volume. bRatings: 0 = Unknown, or information is insufficient. - = Current information suggests inadequate development, little potential for development, little applicability to humans, poor suitability for large populations, poor reproducibility, or poor interpretability. +- = Current information suggests some development, some potential for development, limited applicability to humans, limited suitability for large populations, questionable reproducibility, or questionable interpretability. + = Current information suggests adequate development, potential for further development applicable to humans, suitability for large populations, reproducibility, and interpretability. + + = Current information suggests high development or good potential for substantial development, great applicability to humans, great suitability for large populations, reproducibility, or very good interpretability. SUMMARY Assays of physiologic function in intact subjects are largely markers of response. Few have potential as indicators of expo- sure or susceptibility. Measured charac- teristics often reflect the integrated impact of multiple pathologic altera- tions; they are seldom indicators of speci- fic, single lesions. The respiratory sys- tem responds to injurious agents in only a few ways, so changes in physiologic char- acteristics are seldom specific to causa- tive agents. Many assays are well established and have been used extensively for evaluating patients in the clinic and for studying basic physiologic phenomena. In many cases, therefore, there is information on the relationships among changes in pul- monary function values, subjective sense of illness, and performance disability. Although there is much less information on these relationships for some assays, physiologic assays generally provide a key means of estimating the practical mean- ing of alterations reflected by other types of markers and of estimating the human health impact of environmental exposures. A primary role of the assays, therefore, is to help to determine the extent to which environmental exposures have an impact on health. We have summarized the current clinical assessment of injury to pulmonary endothe- lium and described an example of a biologic .. . . marker of endothelial cell injury that might become useful in either clinical - or screening programs 1n 1umans. Metabolic lung function was chosen as an example of a biologic property under development as a potential marker of lung injury. That choice was intended not to imply that it is expected to be more useful than other potential markers, but rather to illustrate the challenges that must be met in assessing injury to the pulmonary circulation. Indeed, the techniques re- quired to assess this and other biologic markers of endothelial cell injury are cumbersome and require considerable equipment and technical expertise; those are the limitations to their potential application. The need for further valida- tion is also clear. However, the rapid advances in technology that we have wit- nessed in the recent past and others that are probably imminent might, with commit- ment and effort, render some of the tech- niques useful and bring others to light. Intravascular serotonin is transported into endothelium, where it is either se- questered or metabolized by intracellular monoamine oxidase. The resulting metabo- lite appears in the pulmonary venous blood. Angiotensin I is hydrolyzed to angiotensin II by angiotensin-converting enzyme on the luminal cell surface. Exposure to endothelium-damaging toxicants might alter these processes of carrier-mediated uptake, metabolism, and sequestration. Numerous diverse assays are described

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PHYSIOLOGYIN INTACT ORGANISMS in this section, and a tabular summary of their characteristics was thought to be a useful adjunct to the more detailed in- formation in the text. Such tabulation is difficult, because no system of charac- teristics or rating codes fits all the measurements well. If the difficulty and the resulting cautions are appreciated, however, the information in Table 3-3 can provide a useful overview. The definitions 81 of characteristics and codes follow the table. The definitions of characteristics and codes vary from assay to assay and a plus-minus rating is used because it was not considered appropriate to develop a weighted scoring system leading to a sin- gle numeric ranking for each assay. The table is intended as a summary of characteristics.

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