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OCR for page 43
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
.. . . ~
OCR for page 44
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
OCR for page 45
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.
OCR for page 46
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
OCR for page 47
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
OCR for page 48
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
OCR for page 49
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
OCR for page 50
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
OCR for page 51
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
OCR for page 52
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
OCR for page 53
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
OCR for page 72
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.
OCR for page 73
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)
OCR for page 74
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.,
OCR for page 75
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.
OCR for page 76
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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Representative terms from entire chapter:
lung disease
