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Department of Physiology, St. George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, United Kingdom
ABSTRACT
INTRODUCTION
TRACER CLEARANCE IN VIVO
PERMEABILITY COEFFICIENTS
RELATIONSHIP OF CLEARANCE TO PERMEABILITY
COEFFICIENT
CALCULATED THICKNESS OF ASL
MEASURED THICKNESS OF ASL
CONCLUSIONS
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Widdicombe, John. Airway and alveolar permeability and
surface liquid thickness: theory. J. Appl.
Physiol. 82(1): 3-12, 1997.
The thickness of
airway surface liquid (ASL) can be calculated as the ratio of the
permeability coefficient of an absorbed inert tracer to the percentage
rate in which it decreases in content in the airway lumen. The
percentage clearance of radiolabeled diethylenetriaminepentaacetic acid
(DTPA) from human airways or lungs has been measured many times, with a
mean value of 1.04 ± 0.25 (SD) %/min. Rates of clearance from
animal lungs of most species give values of the same order, although
they are lower in the sheep and higher in the dog. Permeability
coefficients have not been measured simultaneously with percentage
clearances and not at all for human tissues. Values for mannitol and
sucrose, of which the former gives a permeability coefficient ~25%
greater than that for sucrose and DTPA in airway tubes and isolated
mucosal sheets from experimental animals, give a mean of ~7.1 × 10
7 cm/s. This corresponds
to thicknesses of ASL of ~20-150 µm for various species. The
assumptions underlying this estimate are discussed. It is concluded
that ASL thickness in vivo may be considerably greater than in vitro
measurements involving rapid freezing of the airway wall. Estimates of
alveolar permeability suggest that either it is very considerably lower
than that of the airway epithelium, that methods to measure alveolar
permeability mainly reflect airway permeability, or both.
airway surface liquid; lung tracer clearance; permeability
coefficient; mucus; mucus thickness; mucociliary transport
INTRODUCTION THERE HAVE BEEN MANY STUDIES of the permeability of the
airway and alveolar epithelia, usually with inert tracers, and the subject has been well reviewed (2, 10, 11, 24, 27, 55). The theory and
basic principles applying to the various methods of assessing
epithelial permeability are well established, and the relationship
between clearance of tracer and airway surface liquid (ASL) volume has
been discussed (9, 18, 35, 46, 98). However, the fact that results from
different methods can be applied to give information about the
thickness of ASL does not seem to have been described before, and this
is the purpose of this review.
The two methods most frequently used to assess permeability are the
rate of loss of tracers from the airway lumen and alveoli into the body
and the determination of permeability coefficients of tracers for the
airway and alveolar epithelia.
TRACER CLEARANCE IN VIVO In humans, the only convenient way of assessing airway and alveoli
epithelial permeabilities is to measure the rate of uptake of a tracer
from the airways and alveoli, with the tracer being administered as an
aerosol. The same method can be used in experimental animals, with the
tracer either given as an aerosol or instilled as a bolus into the
tracheobronchial tree. The tracer usually used is
99mTc-labeled diethylenetriamine
pentaacetic acid (DTPA), a hydrophilic molecule with molecular mass of
492 Da. The rate of uptake from the airways and/or
alveoli is expressed either as the percentage decrease in the pulmonary
tracer content per minute [%clearance of tracer (%Cl)],
as the half time of the initial concentration of the tracer in the
airways and lungs
(t0.5) or,
occasionally but mathematically more correctly, as the exponential
constant [rate constant
(k)] of decay. The three
parameters are related mathematically by simple equations derived from
where
At is the activity at time t and
A0 is the activity at time
0. The relevant equations are
t0.5 = 0.693/k and %Cl = 100 (1
(1)
e0.693/t0.5).
The percent decrease per minute in intraluminal tracer content, %Cl, is the flux of tracer (dQ/dt) divided by the luminal content of tracer (C × V, where C is the luminal concentration of tracer and V is the volume of ASL) times 100, or
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(2) |
PERMEABILITY COEFFICIENTS
The permeability coefficient (P) of a tracer through a tissue such as the airway mucosa is defined as
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(3) |
|
(4) |
C is the concentration difference from lumen to blood or
submucosal tissue.
P values can only be conveniently measured when the luminal concentration of a tracer is controlled and known, so the method is usually applied to epithelial or mucosal sheets in Ussing chambers, to airway tubes in vitro, or to isolated parts of the airways such as the tracheal tube in vivo. The method has not been applied to humans, except in the case of cultured airway epithelial cells in Ussing chambers (25, 112) or isolated small human airways in organ baths in vitro (44).
An alternative approach for experimental animals is to fill the air
spaces of excised lungs during vascular perfusion with liquid
containing a tracer and to measure its rate of loss from the lungs or
uptake into the blood. It is assumed that most of the loss occurs
across the alveolar epithelium, but the alveolar surface area cannot be
measured accurately. Therefore, the method estimates PS = (dQ/dt)/C. P
can be calculated if a value for S is
determined or assumed.
RELATIONSHIP OF CLEARANCE TO PERMEABILITY
COEFFICIENT
|
(5) |
C, so that submucosal concentration of tracer
can be ignored; over short periods (15-60 min), the submucosal
concentration of DTPA is ~3,000 times lower than the luminal one (36,
37), both in vivo and in vitro, so this assumption seems justified.
Thus %Cl varies directly with the permeability coefficient, which is
obvious, and inversely with the thickness of the ASL. The latter is an
important conclusion and may seem surprising. The permeability
coefficient is calculated per unit surface area. The initial flux of
tracer depends on its initial concentration in the ASL, but the
percentage decrease in unit time depends on the size of the reservoir
of tracer, which is directly proportional to ASL thickness per unit
surface area.
Equation 5 has interesting implications. If %Cl is measured and if the value of P is known, we can calculate the thickness of the ASL. Table 1 lists a number of studies measuring %Cl for 99mTc-DTPA in humans. The scatter of values, which is rather small, is presumably due to methodological differences. For example, aerosols of different particle diameter have been used, and in some studies attempts have been made to subtract DTPA clearance due to mucociliary transport or to concentrate deposition of DTPA in large airway or alveolar regions. However, if we ignore values corrected for mucociliary clearance (see below), the mean %Cl is 1.04%/min (range 0.59-1.56 %/min), corresponding to a mean t0.5 of 68 min (range 45-117 min).
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Table 2 gives values of %Cl for species other than humans. The means and ranges are similar to those for humans, with the exception of two species. The dog shows a consistently higher and the sheep a consistently lower value of %Cl than do humans and the other species.
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The permeability of human airway or alveolar epithelium in vivo or in vitro is not known. In experimental animals, the permeability coefficient for DTPA has been measured for the sheep trachea in vivo and the permeability coefficient for DTPA and the coefficients for mannitol and sucrose were measured for a number of in vitro preparations, including airways in organ baths and sheets of airway mucosa or of tracheobronchial epithelium in Ussing chambers.
Table 3 lists some of the results. It omits a few values for species not represented in Table 2 and, therefore, does not permit comparisons, and values for sheets of cultured alveolar or airway epithelial cells, where there is much variation in permeabilities and it is impossible to say how relevant the values are to intact tissues. Nonmammalian species are also omitted. Mannitol (molecular mass 182 Da) and sucrose (molecular mass 360 Da) have been used far more frequently than has DTPA (molecular mass 492 Da) with in vitro experiments. All three tracers are hydrophilic. Based on molecular mass, which is not the only variable to influence permeability, permeabilities would be expected to decrease in the sequence mannitol-sucrose-DTPA. When permeabilities were determined by the same authors for two tracers on the same airway or alveolar preparations, the ratios, giving mannitol as 1.00, are as in Table 4. Values for DTPA and sucrose (in relation to mannitol) are similar and about two-thirds of the permeability for mannitol. Averaging of values for mannitol with those for DTPA and sucrose or using permeability coefficients for mannitol to compare with %Cl based on DTPA may therefore overestimate permeability by up to one-third.
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For tracheal tubes in organ baths in vitro (Table 3), values are
reasonably close, apart from one guinea pig result, which is such an
outlier that it has been excluded from any statistics. For isolated
airway mucosa and airway epithelium in Ussing chambers, the values are
close to those for tracheal tubes. The unweighed mean from Table 3 is
7.63 × 107 cm/s.
CALCULATED THICKNESS OF ASL
Figure 1 is a graphic representation of
Eq. 5, with isopleths for different values of
T. Points are indicated for the mean values for %Cl and P in Tables 1 and
3, for the five species for which both variables have been measured.
The corresponding values for ASL thickness are 53 µm for rats, 46 µm for rabbits, 153 µm for sheep, 27 µm for dogs, and 19 µm for
guinea pigs. In view of the comments above about the permeability for
mannitol compared with sucrose and DTPA, some of these values may be
overestimates by up to one-third.
, Dog; ×,
rat; 
, rabbit; *, guinea pig; 
, sheep. Isopleths are drawn
for airway surface liquid thicknesses with values in µm. Vertical
line corresponds to %Cl for humans and range of
P values for the 5 species included.
Because we do not know the permeability coefficients of human airway mucosa for any of the tracers used, although there are numerous estimates of the value of %Cl for DTPA, we cannot put a point for humans in Fig. 1. However, if the human large-airway permeability is similar to that for the other five species, the possible range of ASL thickness would be indicated by the vertical line in Fig. 1 and would correspond to values from 20 to 58 µm. Again, for reasons given, these could be an overestimate by up to one-third.
The analysis is complicated for a number of reasons.
1) Permeability of the airway mucosa may vary with airway size, and measurements have only been made for trachea and bronchi. The only relevant measurements indicate that permeability is greater in bronchi than in the trachea (32, 88; Table 3), as in %Cl of DTPA (34, 77; Table 2), although this result could be due to thinner ASL in the bronchi. Values for human bronchioles may be higher than for cartilaginous airways (44). ASL thickness will also vary with airway caliber and is smaller in smaller airways (see later). Thus, for all parameters being considered, i.e., %Cl, P, and T, we are dealing with values that may vary along airway length and are usually averaged in calculation.
2) Permeability coefficients have always been measured with liquid-filled preparations, and %Cl measurements have been with air-filled preparations. Although there is no reason to believe that mucosal permeability to 99mTc-DTPA will be different depending on whether the lumen contains air or liquid, there seem to be no published data on this issue. We have recently compared the permeability coefficients for 99mTc-DTPA in liquid-filled and air-filled tracheas in vitro of ferret and rabbit and found no significant difference between the permeabilities in the two conditions (S. Duneclift, U. Wells, and J. G. Widdicombe, unpublished observations).
3) The presence of mucus in the ASL may complicate interpretation of results if the mucus is able to bind to the tracer being used, as is the case with DTPA. Although mucus has a weak affinity for DTPA, it binds it relatively tightly (17). However, the role of any mucus sheet is complicated. The extent to which unbound DTPA can diffuse through the mucus into the epithelium and the extent to which DTPA bound to mucus can subsequently be released for free diffusion are uncertain. The relationship between concentrations of DTPA in the mucus sheet and in the periciliary liquid is not known. It is assumed in this paper that the concentration of free (unbound) DTPA in the mucus gel layer is the same as in the periciliary layer. Although there is no direct evidence on this point, the assumption is consistent with the results of Cheema et al. (17), who measured the uptake of DTPA into airway mucus and showed that the affinity was small, although the binding was tight. If there are different concentrations of free DTPA in the gel and the periciliary fluid, and the former is smaller, and there is free diffusion between the two compartments, than the derived values for total ASL thickness given in this paper would be underestimated. If the estimates of ASL thickness apply mainly to periciliary liquid, because the concentration of free DTPA in the gel is small, then the periciliary liquid will be considerably thicker than the depth of the cilia. This seems unlikely, if only because cilia would have difficulty in working efficiently. In practice, when an aerosol containing the tracer is inhaled, the tracer will first enter any gel or mucus layer, before diffusing into the periciliary liquid. In this sense, the gel may act as a reservoir for the tracer from which it is drawn via the periciliary liquid to cross the epithelial and mucosal barrier. It seems more probable that the estimates of ASL thickness apply to the combination of periciliary liquid and gel layer, the former having a depth close to that of the ciliary length. To resolve this problem, we need more information about the concentrations of free tracer in the mucus compared with periciliary liquid.
If some DTPA binds tightly to mucus, then the bound DTPA will reduce the amount available for diffusion into the mucosa but will not influence the %Cl for transepithelial uptake, since it is independent of concentration. However, in general, any condition that increases mucus secretion and, therefore, the volume and thickness of ASL, should decrease %Cl, even when the tissue permeability is unchanged. For permeability measurements in liquid-filled airway preparations, mucus is unlikely to distort measurements appreciably because of the large pool of liquid in relation to mucus volume and because of the stirring of any potential mucus sheet into the liquid medium.
Studies assessing the component of DTPA clearance due to mucociliary transport and, therefore, bound physically or chemically to mucins (2, 6, 45) have suggested that, for centrally deposited aerosols, mucociliary transport accounts for over one-half DTPA clearance, with the resulting component due to uptake through the epithelium having a %Cl of 0.24-0.68%/min (mean 0.42%/min) (Table 1). This would correspond to a thickness of ASL over two times greater (>40-116 µm in humans) than estimates based on average uncorrected %Cl.
The correction of %Cl for mucociliary transport is based on the calculation of mucociliary transport of an aerosol of albumin, assuming that albumin will behave in the same way as does DTPA and that the larger molecular weight of albumin will prevent its passage through the airway epithelium. However, if mucus binds physically or chemically to DTPA less strongly than to albumin or releases it readily in response to diffusion gradients created by uptake of DTPA through the epithelium, this assumption becomes invalid. It seems probable that the low values of %Cl when corrected for mucociliary transport are in part due to the fact that albumin is cleared by mucociliary transport more readily than is DTPA. For this reason, they have been excluded from the statistical analysis of Table 1. If they are regarded as valid, the calculated ASL thickness will be more than doubled. In addition, the correction assumes that mucus cleared by mucociliary transport is not replaced. This is almost certainly incorrect. If the cleared mucus is replaced by liquid secretions not containing tracer, the concentration would be diluted, and this would contribute to the lower %Cl described.
4) The assumption is made that the volume of aerosol inhaled does not appreciably change the thickness of the ASL. If the surface area of the tracheobronchial tree down to 1-mm-diameter airways is 1,026 cm2 in humans (108), then 1 ml of aerosol liquid, spread uniformly, would increase the thickness of the ASL by only 1 µm. Thus the assumption is probably valid for aerosols but it may be less so for results obtained when liquid is instilled into the airways of experimental animals.
5) Tracer uptake is usually assumed to be a monoexponential relationship with time, although some studies have suggested that with diseased lungs there may be a biexponential clearance (6, 54, 98), with the two components corresponding to healthy and diseased tissues.
6) It is often assumed that delivery
of submicronic aerosol particles containing DTPA gives a measurement of
alveolar, as distinct from tracheobronchial, permeability. Direct
attempts to compare permeabilities assessed by large- and
small-particle aerosols suggest that the latter do give a more rapid
uptake by a factor of ~25% (6, 45), but analysis of all the results in Table 1 shows that there is no correlation between particle size and
%Cl (Fig. 2).
There have been no direct measurements of alveolar permeability in
mammals, although a number of studies on bullfrog lungs give a mean
alveolar permeability of about 2.73 × 107
cm/s (34, 22, 59, 60). The most relevant measurements obtained from
mammals are derived from preparations with perfused vascular beds of
the lungs, with the air spaces filled with liquid containing an
appropriate tracer. Table 5 gives some
results with this preparation. It will be seen that there is
a general consistency in the permeabilities, with the mean being
0.23 × 107
cm/s. This is over thirty times smaller than the permeabilities of
large airways (Table 3).
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These values depend on estimations or assumptions for the alveolar surface area exposed to liquid containing the tracer. This may cause an error, although probably not in a consistent direction and certainly not large enough to explain the 30-fold difference in permeabilities of alveoli and larger airways.
7) It is generally considered that the main barrier for diffusion of hydrophilic agents is at the apical surface of the airway or alveolar epithelial cells, presumably at the tight junctions near to the surface. However, in practice, the barrier may not be at a single anatomical structure. It follows that estimates of ASL thickness will include a component due to the epithelial cells and even deeper tissues. How large this component is has not been determined. However, mechanical removal of the epithelium or its destruction by detergent increases the permeability of the tracheal wall to DTPA in vitro 24-fold (S. Duneclift, U. Wells, and J. G. Widdicombe, unpublished observations) and in vivo 80-fold (111). This supports the view that the epithelium is the main barrier to diffusion of hydrophilic agents and, since the agents cannot pass through the cells, that the tight junctions near the surface are the main part of that barrier.
Calculated permeability coefficients are always based on a measured or assumed surface area of epithelium, such as the area of an Ussing chamber or the internal surface area of an airway tube. It is assumed that DTPA passes through paracellular pathways, but it is impossible to calculate the total area of these pathways. Epithelia may have a greater or smaller degree of convolution or corrugation, and this is difficult to assess. For lipophilic tracers that travel transcellularly, it is impossible to make an accurate estimate of the total cell surface area, including cilia and microvili, through which the agent passes. For this reason, all measurements refer to gross epithelial surface area, as described for DTPA and as is conventional.
8) If
99mTc-DTPA breaks down releasing
99mTc, then the measurements of
%Cl and permeability will be distorted. In most individual experiments, there is no assessment of the extent of possible breakdown, although most studies trying to assess it have indicated that it is not large. For example, measurements of the proportion of
"free" technetium
(99mTcO4)
range from 0.5 to 5%, usually at the lower end (19, 35, 45, 56).
Breakdown might be greater in vivo, when oxygen radicals could cause
release of
99mTcO4.
However, some studies have compared results with
99mTc-DTPA and
113mIn-DTPA or
14C-DTPA, the latter two molecules
being far more resistant to breakdown. Similar results were obtained
between 99mTc and the other
isotopes (17, 56, 74). If there were to be appreciable breakdown of
99mTc-DTPA, one would expect a bi-
or multiexponential decay in radioactivity, and this has never been
described for healthy lungs, although it may be present in disease. In
the latter case, it is assumed that the multiexponential relationship
is due to pathophysiological variations rather than to the breakdown of
99mTc-DTPA. Furthermore, the fact
that the published values for %Cl and for permeability in individual
species show relatively small variance (e.g., Tables 1-4),
suggests that either the breakdown is uniform across the field of
research or that it is negligible. Mannitol and sucrose are labeled
with 14C, and the leaching of this
element from the sugars is thought to be insignificantly small. The
fact that the measurements with mannitol and sucrose give results close
to those with DTPA, allowing for differences in molecular weight, may
support the view that breakdown of
99mTc-DTPA is small.
9) The creation of unstirred layers for tracers diffusing through the airway or alveolar epithelium would complicate the calculations in this and most other papers. However, both theoretical calculations and measurements suggest that for molecules such as DTPA, mannitol, and sucrose, which have low permeability coefficients because they are hydrophilic and can only pass through paracellular pathways, substantial unstirred layers are unlikely and that they would not significantly distort the calculations of permeability coefficients (3, 106).
MEASURED THICKNESS OF ASL
Early measurements of the thickness of ASL in the tracheobronchial tree gave values on the order of 15-30 µm (105). These estimates were based on histological light-microscopic studies, and the thickness given includes both the periciliary layer and any superimposed gel on top. The structural appearance of the ASL suggests that the gel is thicker than the periciliary liquid and that the cilia reach up to touch it. Recent measurements of the total thickness of ASL, without distinction between periciliary liquid and mucus sheet, give values of 33 µm (96) for the sheep trachea in vitro and 100-200 µm for guinea pig trachea in vitro and in vivo (86, 97). However, electron-microscopic studies using rapid-freezing methods on frog palate and monkey and bovine airways give values as small as 1 µm or less for ASL lying above the cilia (26, 85, 114). Including ciliary depth, these measurements suggest that the total ASL may be only 5-10 µm thick. The reason for the discrepancy in values may be because, in some preparations, there are sheets or plaques of mucus secreted from submucosal glands that overlie the periciliary fluid and that these gel structures are absent from the electron-microscopic studies (105). It may be significant that conventional histological methods with the rat and rabbit, species that have no or few submucosal glands, gave values of 0.1 µm for bronchioles and 5 µm above the cilia for large airways (93, 116). Stimulation of gland secretion by methacholine increases the total thickness of bovine ASL (assessed by election microscopy) from 6.9 to 27.9 µm (114), a value not far from those calculated in this paper, although a similar effect was not seen in the sheep (96).
Electron-microscopic estimates of the thickness of the alveolar lining
substance give average values of ~0.1-0.24 µm (5, 109),
compared with a value of 0.15 µm obtained from physiological measurements (99). Measurements of alveolar permeability coefficients of liquid-filled lungs give values one-thirtieth of those of the trachea and bronchi (Table 5). If we assume that permeability coefficient for the alveoli is 0.23 × 107 cm/s, then the %Cl for
a 0.15-µm-thick layer would be 9.2%/min. This is nine times higher
than the values estimated by uptake of submicronic aerosols of DTPA
(0.59-1.56%/min, Table 1). Either the permeability of the
alveolar epithelium must be much smaller than estimated from Table 5,
although this is already 30 times smaller than values for large
airways, or the majority of the aerosol is being deposited in
conducting airways where the ASL is thicker. The latter seems more
likely. The possibility that aerosol deposited in the alveoli greatly
increases the thickness of the lining layer (and therefore decreases
%Cl) is unlikely. One milliliter of liquid spread over 100 m2 surface area would increase the
lining liquid thickness by only 0.01 µm.
A further consideration is that submicronic aerosol particles may not spread over the whole alveolar surface. Calculations (35) suggest that they may only occupy 1/770 (<0.1%) of the surface. If they do not mix with hydrophobic surface-lining liquid, they might cause localized projections up to 1 µm thick. Absorption of DTPA from these projections, expressed as %Cl, might be four times slower than had the aerosol spread out uniformly over the alveolar surface, since effective ASL thickness due to the projection is greater. A fourfold increase in the number of submicronic aerosol particles inhaled results in a 24% increase in DTPA clearance (35), suggesting that a spreading of the aerosol may be important, although probably not very much.
CONCLUSIONS
1) Estimates of airway ASL thickness based on tracer clearances and permeability coefficients give values higher than recent electron-microscopic measurements. The difference may be explained by the presence or absence of gellike sheets or plaques of mucus secreted from submucosal glands.
2) Differences in %Cl need not reflect differences in permeability but could be due to changes in thickness of ASL. This may be especially important in some physiological conditions and in lung diseases. For example, several studies have shown that %Cl is increased when the lungs are inflated or during positive end-expiratory pressure ventilation (21, 50, 65, 67, 73, 75, 79). This result is usually interpreted as due to a thinning of the epithelium with an increase in its permeability or to recruitment of previously closed alveoli. However, a thinning of the ASL could also contribute to the result. Similarly, %Cl may give a normal value, even if there is increased permeability of the airway epithelium, if ASL thickness is also increased, for example, by mucus secretion.
3) Attempts have been made to compare tracheobronchial with alveolar clearance, usually based on use of aerosols of different particle size. Although the thickness of the alveolar surface liquid has never been directly measured, electron-microscopic and physiological studies suggest that it is only a fraction of a micrometer. It follows that extremely high clearance values should be obtained due to the small liquid thickness. Because these values have not been reported, either the alveolar epithelium permeability is very low, most of the aerosol is being deposited in the airways, or both.
4) It should be possible to devise methods, at least in experimental models, where measurements of %Cl and permeability in the same preparation provide a reasonably accurate measurement of ASL thickness.
ACKNOWLEDGEMENTS
I am greatly indebted to Dr. T. Okubo (Yokahama, Japan) for stimulating discussion that led to many of the ideas in this paper. I am also grateful to Drs. Paul Richardson, Edith Puchelle, Daffid Walters, and Ursula Wells for valuable discussions.
FOOTNOTES
Address for reprint requests: J. G. Widdicombe, Sherrington School of Physiology, UMDS, St. Thomas' Hospital, Lambeth Palace Rd., London SE1 7EH, UK.
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