Vol. 87, Issue 5, 1831-1842, November 1999
Pulmonary inflammation alters the lung disposition of
lipophilic amine indicators
Said H.
Audi1,2,
David L.
Roerig4,5,
Susan B.
Ahlf5,
Win
Lin2, and
Christopher A.
Dawson1,2,3,5
1 Biomedical Engineering
Department, Marquette University, Milwaukee 53201-1881;
2 Departments of Pulmonary
Medicine and Critical Care,
3 Physiology, and
4 Anesthesiology and
Pharmacology/Toxicology, Medical College of Wisconsin, Milwaukee
53226; and 5 Zablocki Veterans
Affairs Medical Center, Milwaukee, Wisconsin 53295
 |
ABSTRACT |
Many lipophilic amine compounds are rapidly
extracted from the blood on passage through the pulmonary circulation.
The extent of their extraction in normal lungs depends on their
physical-chemical properties, which affect their degree of ionization,
lipophilicity, and propensity for interacting with blood and tissue
constituents. The hypothesis of the present study was that changes in
the tissue composition that occur during pulmonary inflammation would
have a differential effect on the pulmonary extraction of lipophilic amines having different properties. If so, measurement of the extraction patterns for a group of lipophilic amines, having different physical-chemical properties, might provide a means for detecting and
identifying lung tissue abnormalities. To evaluate this hypothesis, we
measured the pulmonary extraction patterns for four lipophilic amines,
[14C]diazepam,
[3H]alfentanil,
[14C]lidocaine, and
[14C]codeine, along
with two hydrophilic compounds,
3HOH and
[14C]phenylethylamine,
after the bolus injection of these indicators into the pulmonary artery
of isolated lungs from normal rabbits and from rabbits with pulmonary
inflammation induced by an intravenous injection of complete Freund's
adjuvant. The pulmonary extraction patterns, parameterized using a
previously developed mathematical model, were, in fact, differentially
altered by the inflammatory response. For example, the tissue
sequestration rate, kseq (ml/s), per unit
3HOH accessible extravascular lung
water volume significantly increased for diazepam and lidocaine, but
not for codeine and alfentanil. The results are consistent with the
above hypothesis and suggest the potential for using lipophilic amines
as indicators for detection and quantification of changes in lung
tissue composition associated with lung injury and disease.
diazepam; lidocaine; alfentanil; codeine; multiple-indicator
dilution; phenylethylamine; mathematical modeling
| |
INTRODUCTION |
RECENTLY, WE EXAMINED the pulmonary disposition of a
group of lipophilic amine compounds having differential extraction
patterns with use of the multiple-indicator dilution (MID) method (1). We developed a mathematical modeling approach for parameterizing the
extraction patterns of the resulting MID data (1, 47). One objective of
those studies was to provide the tools necessary for examining the
hypothesis that a change in lung tissue composition can be detected by
a change in the extraction patterns of compounds having different
physical-chemical properties. This implies their potential use for
detecting and quantifying disease-related changes in lung tissue
composition (1, 10, 12, 27, 36, 37, 39, 41, 43-47). The specific
objective of the present study was to determine the impact of a
pulmonary inflammatory response on the pulmonary extraction of four
lipophilic amine test indicators: [14C]diazepam,
[3H]alfentanil,
[14C]lidocaine, and
[3H]codeine. These
compounds, which have a range of physical-chemical properties as
reflected by differences in
pKa,
lipophilicity, and affinity to plasma proteins (14, 29, 44, 45) given in Table 1, were chosen
because they are sufficiently and differentially extracted during a
single pass through the pulmonary circulation of normal lungs for MID
quantification (1, 3, 12, 45, 47). The studies were carried out on
lungs isolated from normal rabbits and from rabbits treated by
intravenous injection of complete Freund's adjuvant (CFA), which
produces a well-characterized inflammatory response in the rabbit lung
dominated by a significant increase in macrophages, histiocytes, and
granulomata (7, 13, 38, 47). On the basis of these and other
characteristics, this animal model has been considered useful for
elucidating mechanisms that may be relevant to human interstitial lung
diseases (13). The MID data were parameterized using a previously
developed kinetic model and methodology (1, 47). The results indicate
that the pulmonary extraction patterns for the chosen group of
lipophilic amines and the resulting parameters were differentially
altered by the inflammatory response, supporting the hypothesis that
the extraction patterns of lipophilic amines may provide a useful signature of lung tissue composition (1, 12, 27, 37, 41, 43, 47).
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Table 1.
Descriptors of properties of lipophilic amine compounds thought to be
important in determining their lung disposition
|
|
Glossary
R, F, and T
| A-aDO2 |
Difference between alveolar and arterial
PO2
|
| CD(t) |
Venous effluent concentration of test indicator at
time t after bolus injection
|
| CFA |
Complete Freund's adjuvant
|
| CF(t) |
Venous effluent concentration of
3HOH at time
t after bolus injection
|
| CR(t) |
Venous effluent concentration of vascular reference indicator FITC-Dex
at time t after bolus injection
|
| CT(t) |
Tubing outflow curve
|
| F |
Flow
|
| FITC-Dex |
Fluorescein isothiocyanate-labeled 40,000-mol wt dextran
|
 c(t) |
Transit time distribution that accounts for effects of capillary
transit time distribution on indicator extraction pattern
|
| kseq |
Sequestration rate
|
| MID |
Multiple-indicator dilution
|
| Pa |
Arterial pressure
|
| PEA |
Phenylethylamine
|
| PS |
Permeability-surface area product
|
| Pv |
Venous pressure
|
| Qc |
Capillary volume
|
| QF |
Flow-limited volume accessible to PEA
|
| QR |
Virtual volume reflective of capacity of class of rapidly equilibrating
amine-tissue interactions
|
| QS |
Virtual volume reflective of capacity of class of slowly equilibrating
amine-tissue interactions
|
| Qti |
Tissue volume
|
| QV |
Vascular volume
|
| QW |
Extravascular water volume
|
| RDv |
Vascular relative dispersion
|
| Res(t) |
Fraction of injected test indicator in lung tissue at a given
time t after bolus injection
|
| R, F, and T |
Mean transit times of
CR(t),
CF(t),
and
CT(t),
respectively
|
| VF |
Virtual volume, which includes QF
and effects of rapidly equilibrating associations of PEA within
QF
|
| z score |
Normal deviate
|
 2R and 2T |
Second central moments of
CR(t)
and
CT(t),
respectively
|
 |
Mean sojourn time, i.e., mean time lipophilic amine molecules
associated with slowly equilibrating class of associations spend in
lung tissue before they return to capillary (in s)
|
| |
EXPERIMENTAL METHODS |
The studies were performed on 25 New Zealand White rabbits of either
gender [2.70 ± 0.29 (SD) kg]. Sixteen of the rabbits (2.56 ± 0.22 kg) were each given a 1-ml ear vein injection of CFA
(8.5 ml Bayol F, 1.5 ml Arlacel, and 5 mg
Mycobacterium butyricum) (7, 13, 38,
47). The remaining nine rabbits (2.79 ± 0.27 kg) served as
controls. Vehicle control experiments were not performed, because the
objective was to produce changes in lung tissue composition and not to
study the CFA-induced inflammatory response per se. At 3-98 days
after the CFA or no treatment, the rabbits were anesthetized and the
MID studies described below were carried out on the lungs. Just before
the administration of anesthesia in 17 of the 25 rabbits studied, a
1-ml blood sample was obtained from an ear artery for arterial blood
gas analysis and for the estimation of the difference between the
alveolar and arterial PO2:
A-aDO2 = (PIO2 
PaCO2/0.8) PaO2, where
PIO2
is inspired PO2 and
PaCO2 is arterial
PCO2, and
PaO2 is arterial
PO2.
Isolated Rabbit Lung Preparation
As previously described (1, 3, 47), each of the rabbits was given
chlorpromazine hydrochloride (25 mg/kg im) followed by pentobarbital
sodium (20-25 mg/kg) via an ear vein and then heparinized (1,200 IU/kg) and exsanguinated via a carotid artery catheter. The pulmonary
artery, vein, and trachea were cannulated. The lungs were removed from
the chest and attached to the perfusion system primed with a
physiological salt solution (in g/l: 0.35 KCl, 0.37 CaCl2 · 2 H2O, 0.29 MgSO4 · 7 H2O, 0.16 KH2PO4,
6.9 NaCl, 1 glucose, 2.1 NaHCO3,
45 BSA) (1, 3, 47). The perfusion system included a heated perfusate
reservoir and a Master Flex roller pump, which pumped perfusate at a
constant mean flow (F) of 3.33 ml/s from the reservoir into the
pulmonary artery, with the left atrial pressure set equal to
atmospheric (pleural) pressure by adjustment of the height of the
venous outflow into the recirculation reservoir. Arterial
(Pa) and venous
(Pv) pressures were referenced to the level of the left atrium. The lung was ventilated with 95%
O2-5%
CO2 at 10 breaths/min under
positive pressure with use of a solenoid respirator with
end-inspiratory and end-expiratory airway pressures of 7.12 ± 0.42 and 1.44 ± 0.47 (SD)
cmH2O, respectively. The perfusate
was equilibrated with the respiratory gas mixture, which maintained the
pH at 7.39 ± 0.06 (SD) at 37°C. Before each of the bolus
injections described below, the ventilator was stopped at end
expiration for the duration of the sampling period.
To produce a bolus injection, a solenoid-operated injection loop (1, 3,
47) was situated in the inflow tubing so that a 1.0-ml bolus could be
introduced into the inflow stream without changing the flow or
pressure. Just before injection, the venous outflow was directed into
the sample tubes of a modified (1, 3, 47) Gilson Escargot fraction
collector. One hundred 2-ml samples were collected with a sampling
interval of 0.6 s.
Bolus Composition
The 1.0-ml bolus of the perfusate solution contained 2.5 mg of
fluorescein isothiocyanate-labeled 40,000-mol wt dextran (FITC-Dex) and
0.5 µCi of 3H or 0.1 µCi of
14C of one or more of
[14C]diazepam,
[3H]alfentanil,
[14C]lidocaine,
[3H]codeine,
3HOH, or
[14C]phenylethylamine
(PEA). The latter two hydrophilic indicators were included as test
indicators to trace changes in the perfused extravascular water volume
and perfused endothelial surface (47), respectively. The specific
activities for
[14C]diazepam,
[3H]alfentanil,
[14C]lidocaine,
[3H]codeine,
3HOH, and
[14C]PEA were 55, 340, 56, 40, 50, and 55 mCi/mmol, respectively.
After each experiment, the lungs were removed from the perfusion
system, and an additional bolus containing FITC-Dex was perfused, with
the arterial and venous cannulas connected directly together. The data
from this injection were used to obtain the tubing concentration vs.
time curve
[CT(t)]
and the moments thereof for the passage of the bolus through the tubing
from injection to fraction collector in the absence of the lungs.
The concentration of the FITC-Dex in the outflow samples was measured
spectrophometrically (494 nm). The
14C and/or
3H was measured by liquid
scintillation counting. Measured quantities of the injectate solution
were added to sample tubes collected before the emergence of the
indicators. These samples served as internal standards for the
calculation of indicator concentrations. For lungs from CFA-treated and
normal animals, the fractions of the injected FITC-Dex and
3HOH recovered in the collected
samples, calculated on the basis of the internal standards, were 95.8 ± 3.9 and 98.8 ± 3.4% (SD), respectively. The fractions of
14C injected as PEA recovered in
the collected samples from normal and CFA-treated lungs were 84.7 ± 2.25 and 64.1 ± 9.6%, respectively. The fractions of the injected
lipophilic amines recovered in the collected samples from normal lungs
were 99.2 ± 2.0% (SD) for alfentanil, 94.4 ± 2.3% for
diazepam, 91.5 ± 3.8% for lidocaine, and 88.8 ± 3.2% for
codeine. These fractions were generally lower in lungs from CFA-treated
animals, as can be seen in Fig.
1, where the fractions of the
injected amines remaining in the lung at the end of the 60-s sampling
period are plotted vs. time after CFA treatment.
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Fig. 1.
Percentage of designated compounds remaining in lung at end of 60-s
sampling period. CFA, complete Freund's adjuvant.
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Caspase-3 Assay
After each experiment, the lungs were weighed and then lyophilized to
determine the ratio of wet to dry weight. The lyophilized lungs were
then ground to a powder with a glass mortar and pestle. The dried lung
powder was used to assay for caspase-3 activity, as an index of lung
inflammation (18), with use of a kit developed by Pharmingen (San
Diego, CA) as follows. The dried lung powder (200 mg) was homogenized
with 4.0 ml of the lysis buffer for 2 min with a Bio-Homogenizer
(Biospec Products, Barthesville, OK) at its highest speed. Homogenates
were kept at 4°C or frozen until assayed. The lysis buffer
consisted of 10 mM Tris, 10 mM
NaH2PO4/Na2HPO4, 130 mM NaCl, 10 mM sodium pyrophosphate, and 1% Triton X-100 at pH
7.5. The assay buffer was pH 7.5 PBS. For each assay, 100 µl of
appropriately diluted homogenate were mixed with 900 µl of PBS and 10 µl (10 µg) of the fluorogenic tetrapeptide caspase substrate
N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin
with and without 10 µl (1 µg) of the caspase-3 inhibitor
N-acetyl-Asp-Glu-Val-Asp-CHO. The
reaction mixtures were incubated at 37°C for 1 h, then the fluorescence was measured on a spectrophotofluorometer (model 650-10S,
Perkin-Elmer) with use of a 1 × 1 cm cuvette. Reaction mixtures
without the substrate served as negative controls. All lung homogenates
were assayed in duplicate, and the coefficient of variation was 9.4%.
Caspase-3 activity is expressed as the difference in fluorescence units
(in mV) between reaction mixtures with and without the inhibitor.
Histological Studies
After perfusion, one lung from a normal rabbit and one lung from a
rabbit 14 days after CFA treatment were fixed in 10% neutral Formalin,
and 5-µm-thick sections were stained with standard hematoxylin-eosin.
| |
EXPERIMENTAL RESULTS |
Values of the three indexes of lung inflammation, namely, the in vivo
A-aDO2, lung
wet weight, and caspase-3 activity over the time course of the
inflammatory response, are given in Fig. 2.
By these measures, the injury phase of the inflammatory response peaked
at ~1-2 wk after CFA treatment, then the inflamed lungs entered
a recovery phase characterized by the return of these indexes toward
normal values. Nine CFA-treated rabbits were studied within the 1- to
2-wk period after CFA treatment. In what follows, the parameter values
from this group will be averaged and referred to as being obtained from
the "peak response."
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Fig. 2.
Lung wet weight, alveolar-arterial
PO2 difference
(A-aDO2), and
caspase-3 activity per gram of lung dry weight vs. time after CFA
treatment.
|
|
Figure 3 shows histological sections of
normal lungs and lungs treated with CFA for 14 days. Histological
sections of the CFA-treated lungs show diffuse interstitial infiltrate
primarily composed of epithelioid histiocytes with fewer lymphocytes,
plasma cells, and eosinophils and, rarely, neutrophils. Some of the
histiocytes are multinucleated, forming giant cell granulomatous
patterns. Alveolar spaces contain many macrophages and some
inflammatory cells. There is no evidence of vasculitis. The lung wet
weight and microscopic changes due to the inflammatory response
observed in the present study are consistent with those reported in
previous studies of this model of pulmonary inflammation (7, 13, 38, 47).
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Fig. 3.
Tissue sections from normal lung (A
and B) and lung treated for 14 days
with CFA (C and
D). Tissue sections were stained
with hematoxylin-eosin. Original magnification ×40 for
A and
C and ×100 for
B and
D.
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|
The CFA-treated lung wet-to-dry weight ratios [5.27 ± 0.33 (SD)] were equal to or lower than those for normal
lungs (5.79 ± 0.1), which is consistent with lung wet weight
changes due to changes in cellular composition in this model of
pulmonary inflammation (7, 13, 38, 47) rather than to edema.
The pulmonary arterial-venous pressure difference was significantly
(P < 0.001) higher in peak-response
lungs (10.6 ± 1.9 cmH2O) than
in normal lungs (6.1 ± 0.7 cmH2O) with the same perfusate flow of 3.33 ml/s. The pulmonary arterial-venous pressure difference returned toward the normal value during the recovery phase of the
inflammatory response.
The in vivo arterial blood pH and
PCO2 were 7.46 ± 0.05 and 31.4 ± 2.2 (SD) Torr, respectively, for the peak-response rabbits
compared with 7.40 ± 0.09 and 26.3 ± 5.0 Torr, respectively, for the normal rabbits, but the differences were not statistically significant.
MID Results
Concentration vs. time data.
Figure 4 exemplifies the venous effluent
concentration vs. time curves for FITC-Dex,
[14C]diazepam,
[3H]alfentanil,
[14C]lidocaine,
[3H]codeine,
3HOH, and
[14C]PEA from normal
and CFA-treated lungs at different stages of the inflammatory response.
The patterns of the test indicator curves relative to those for the
vascular reference indicator and to each other changed over the time
course of the inflammatory response. For example, with diazepam there
was a substantial reduction in its recovery and a leftward shift in the
peak of its outflow concentration curve relative to that for the
FITC-Dex. Comparison of lidocaine and codeine concentration curves
(Fig. 4B) reveals that CFA treatment
had a greater effect on the extraction of lidocaine than of codeine.
The differences can be appreciated by noting the reversal of the order
of the relative magnitudes of their respective concentration curves.
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Fig. 4.
Examples of venous effluent concentration vs. time curves for
fluorescein isothiocyanate-labeled dextran (FITC-Dex),
[14C]diazepam,
[3H]alfentanil,
[14C]lidocaine,
[3H]codeine,
3HOH, and
[14C]phenylethylamine
(PEA) after bolus injection of these compounds into pulmonary artery of
isolated perfused lungs from normal and CFA-treated rabbits at
different stages of inflammatory response (days after CFA treatment).
Solid lines, model fits as described in DATA
ANALYSIS.
|
|
During the injury phase of the inflammatory response, there was a
progressive reduction in the peak and a prolongation in the
3HOH concentration curve relative
to that from normal lungs, reflecting the increase in the lung water
volume (Fig. 4C). The effects of the
inflammatory response on the PEA curve were relatively small, although
the tail of the PEA concentration vs. time outflow curve tended to be
depressed during the injury phase. All these changes in the
concentration curves had nearly disappeared by 98 days after CFA treatment.
Residue curves.
Figure 4 shows that the FITC-Dex outflow concentration curve also
changed over the time course of the inflammatory response. This is the
result of increased vascular transit time heterogeneity in CFA-treated
lungs expressed by the relative dispersion
(RDV) of vascular transit times
(Table 2), where
RDV is the square root of the
second central moment (variance) of lung vascular transit times divided
by the lung vascular mean transit time (see Eq. 4). Thus it is useful to separate the effects of CFA
treatment common to reference and test indicators from those unique to
the test indicators by transforming the venous effluent concentration data of the vascular
[CR(t)]
and test
[CD(t)]
indicators in Fig. 4 into the residue curves shown in Fig.
5 by use of the following relationship
![Res(<IT>t</IT>) = F <LIM><OP>∫</OP><LL>0</LL><UL><IT>t</IT></UL></LIM> [C<SUB>R</SUB>(<IT>t</IT>) − C<SUB>D</SUB>(<IT>t</IT>)] d<IT>t</IT>](/content/vol87/issue5/fulltext/1831/img002.gif)
|
(1)
|
where,
for a given test indicator, Res(t)
is interpreted as the fraction of the injected test indicator that is
in the lung tissue at a given time t
after the bolus injection.
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Fig. 5.
Example of residue functions
[Res(t)] for
[14C]diazepam,
[3H]alfentanil,
[14C]lidocaine, and
[3H]codeine at
different stages of inflammatory response (days after CFA treatment)
obtained from corresponding concentration data shown in Fig.
4.
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|
The differences in the CFA-induced changes in the extraction patterns
of the four lipophilic amines described above with respect to Fig. 4
are emphasized by this transformation, such that one can more readily
appreciate the CFA-induced changes in the pattern formed by the
combination of the four lipophilic amines.
To produce a concise parameterization of these patterns for statistical
analysis and potentially for use in pattern recognition algorithms, we
used the following analysis developed previously (1, 47).
| |
DATA ANALYSIS |
FITC-Dex and 3HOH
The mean transit times () and the
second (2) and third
(m3) central moments of the
outflow curves of FITC-Dex
[CR(t)]
and 3HOH
[CF(t)]
and
CT(t)
were obtained by fitting each to a shifted random walk function, the
functional form of which can be specified by its first three moments
(1, 2, 47).
The vascular volume (QV) and the
perfused extravascular water volume
(QW) were estimated from
|
(2)
|
|
(3)
|
where
F is flow and
R,
F, and
T are the
mean transit times of
CR(t),
CF(t),
and
CT(t), respectively.
The vascular relative dispersion
(RDV) was estimated from the
moments of
CR(t)
and
CT(t)
from
|
(4)
|
where
2R and
2T are the second central moments of
CR(t)
and
CT(t), respectively.
Lipophilic Amine Compounds
For each of the four lipophilic amine test indicators studied,
parameter estimation was carried out using a previously described model
(1, 47). Briefly, each model capillary element is composed of a
capillary volume (Qc) and a
tissue volume (Qti). The model assumes that equilibration between the free and protein-bound test
indicator (lipophilic amine) in Qc
(1, 3, 12, 47) is rapid and that the free form of the test indicator is
the species having diffusional access to
Qti. Within
Qti, multiple classes of
amine-tissue associations are represented with different dissociation rate constants (1, 47), which, along with the amine associations within
Qc, are represented by the
following parameters: QR (ml), QS (ml), mean sojurn time
(, s), and sequestration rate (kseq, ml/s) (1,
47). Each of these parameters can involve several kinetic parameters
that are not separately identifiable (1, 47). Physically,
QR and
QS represent the capacities of two
classes of amine-tissue associations referred to as rapidly and slowly
equilibrating classes, respectively. The rapidly (relative to the
capillary mean transit time) equilibrating amine associations within
Qti and
Qc, which are not mathematically
distinguishable from each other, are represented by
QR. The slowly equilibrating amine-tissue associations within
Qti are quantified by
QS and by
, which is the mean time the lipophilic
amine molecules associated with the slowly equilibrating class of
associations spend in the lung tissue before returning to the capillary
(1, 47). A third class of amine-tissue associations with dissociation rate constants that are so small that there is virtually no return to
the perfusate within the MID sampling period is described by kseq.
[14C]PEA
The model used to interpret the uptake of PEA by the pulmonary
endothelial cells was developed previously (47). The model assumes that
PEA has access to a flow-limited volume
(QF), within which it can
participate in rapidly equilibrating associations with the tissue, or
it can be transported into the endothelial cells via a linear
unidirectional transport mechanism having a permeability-surface area
product (PS) (47). The model
parameters are then PS (ml/s) and
VF (ml), a virtual volume, which
includes QF and the effects of the
rapidly equilibrating associations of PEA within
QF (47).
PEA is metabolized within the pulmonary endothelial cells to
phenylethylacetic acid (PAA) (16, 47). Thus, as time progresses during
bolus passage, a fraction of the
14C injected as
[14C]PEA returns to
the perfusate as
[14C]PAA. Previously
(47), we found that, after the bolus injection of
[14C]PEA into the
pulmonary artery of isolated perfused normal or CFA-treated lungs at
the flow used in the present study,
[14C]PAA was not
detectable in the 14C outflow
curve until samples collected after the peak of the FITC-Dex
concentration vs. time curve. Thus, as described previously (47), the
kinetic model parameters descriptive of PEA-tissue associations,
namely, PS and
VF, were obtained by fitting the PEA model to the
[14C]PEA concentration
curve, only up to the peak of the FITC-Dex concentration curve.
Numerical aspects of parameter estimation were carried out as
previously described using 3HOH
concentration data to obtain the function
c(t), which accounts for the effects of the capillary transit time
distribution on the indicator extraction pattern (2, 47). Model fits to the data are exemplified in Fig. 4. The overall coefficients of variation for the model fits were on average 8.0 ± 0.5, 5.9 ± 0.3, 9.9 ± 0.6, 10.1 ± 0.4, and 3.0 ± 0.4% (SE)
for diazepam, alfentanil, lidocaine, codeine, and PEA, respectively.
| |
MODEL RESULTS |
Normal vs. Peak Response
The calculated MID parameters for the four lipophilic amine test
indicators studied and the other MID parameters estimated from normal
and peak-response lungs are given in Tables 2 and 3.
The estimated values of QV and the
PS for PEA from peak-response lungs
were not different from those estimated from normal lungs. However,
RDV, the extravascular water
volume accessible to 3HOH
(QW), and the virtual volume
accessible to PEA (VF) were
higher in peak-response than in normal lungs. The ratio of
extravascular water volume accessible to
3HOH
(QW) to the gravimetrically
measured lung water volume decreased from 0.79 ± 0.02 (SE) in
normal lungs to 0.42 ± 0.03 in peak-response lungs.
For the four lipophilic amine test indicators, the differential effects
of CFA treatment on the extraction patterns are revealed in the
parameter patterns shown in Table 3.
QR,
QS, and
kseq are
extensive parameters reflecting changes in the total number of sites of
interactions between the lipophilic amine test indicators and the lung
tissue (1, 47). This number might be affected by a change in the lung
tissue mass, a change in the fraction of the lung tissue mass
accessible to the test indicators via the perfused vascular volume,
and/or a change in tissue composition, i.e., in the number of sites per
unit mass. The PEA PS and
QW are indexes of the perfused
vascular surface area and extravascular volume accessible via the
perfusate, respectively (47). Thus the normalization of
QR,
QS, and
kseq to
PS or
QW can provide additional
information as to whether parameter changes shown in Table 3 simply
reflect changes in lung tissue mass, changes in the fraction of the
mass that is being perfused, or changes in the lung tissue composition.
Because the PS was hardly affected by
the CFA treatment, normalization to PS
is not reported. However, normalization to
QW, which increased substantially
during the inflammatory response, is provided in Table
4.
Changes Over the Time Course of the Inflammatory Response
In addition to the 1- to 2-wk studies, lungs were studied at different
times after CFA treatment to confirm the inflammation-recovery cycle
typically observed in the rabbit CFA model (7, 13, 38). It also
provided a gradation in the response, allowing for correlations between
measured indexes of inflammation and the model parameters.
For the lipophilic amines studied, the estimated values of the MID
parameters reflected the differences in the extraction patterns of the
various amines at the different stages of the inflammatory response.
For instance, Fig. 6 shows that the
estimated values of QS, the
virtual volume reflective of the slowly equilibrating amine-tissue
interactions, were highly correlated with lung wet weight for diazepam,
but not for codeine. In addition, the hysteresis in the plot of
kseq for
lidocaine vs. lung wet weight (Fig. 7) reveals that, for a given lung wet weight,
kseq for
lidocaine during the injury phase was lower than that during the
recovery phase of the inflammatory response. This result suggests that kseq for
lidocaine is not simply a measure of lung wet weight and that other
more specific aspects of lung composition may be responsible for the
changes in the pulmonary disposition of lidocaine at the different
stages of the inflammatory response. For each of the four lipophilic
amine compounds studied, measures of correlation between the estimated
values of the MID parameters and lung wet weight over the time course
of the inflammatory response are given in Table
5.
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Fig. 6.
Relationship between lung wet weight and
QS for diazepam (solid symbols)
and codeine (open symbols) in lungs from normal (triangles) and
CFA-treated (circles) rabbits. Range in lung wet weight for CFA-treated
rabbits was obtained by studying animals at different time points after
3-98 days of CFA treatment. Coefficients of determination
(r2) were 0.09 and 0.79 for codeine and diazepam, respectively.
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Fig. 7.
Relationship between lung wet weight and sequestration rate
(kseq) for
lidocaine in lungs from normal ( ) and CFA-treated rabbits during
injury ( ) and recovery ( ) phases of inflammatory response. Phases
are defined by whether lungs were increasing (injury phase) or
decreasing (recovery phase) in wet weight, as indicated by direction of
arrows superimposed on lines connecting points. For consecutive days
after CFA treatment, there was significant
(P < 0.05) hysteresis in
relationship, as indicated by runs test (40) applied to residuals about
linear regression line through data ranked by days after CFA
treatment.
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Table 5.
Coefficient of determination for correlation between MID parameters and
lung wet weight for lungs from CFA-treated rabbits
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To determine whether the changes in the parameters for a given
lipophilic amine and among the different lipophilic amines studied at
the different stages of the inflammatory response were significant,
each individual parameter was transformed into a z score, i.e., (individual parameter normal group mean for that parameter)/standard deviation of
normal group parameter (47, 49), also referred to as the normal deviate
(49). This allows for a statistical evaluation of the probability that
a particular parameter value for a given individual lung falls within
the distribution of that parameter in the normal lungs (49). In other
words, the probability that an individual parameter belongs to the
control distribution and, hence, is not significantly different from
that in normal lungs, is <1% when its
z score exceeds |3| (49).
The changes in the extraction patterns of the lipophilic amines at the
different stages of the inflammatory response exemplified in Figs. 4
and 5 are reflected in the calculated
z scores of the parameters shown in
Fig. 8. To put the individual values in
perspective, z scores of ±3 are
designated by the horizontal dashed lines. From Fig. 8 it is clear that
certain parameters and different parameters for different test
indicators track the time course of the inflammatory response better
than others. The z scores for
kseq for all four
lipophilic amines studied, QS for
diazepam, alfentanil, and lidocaine, and
QR for lidocaine were consistently >3 from 3 to 28 days after CFA treatment.
QS for codeine,
QR for diazepam, alfentanil, and
codeine, and for all four lipophilic amines were not discriminating parameters. All lungs from 3 to 48 days
after CFA treatment were identifiable as such by
z scores exceeding |3| for
at least five of the eight discriminating parameters. In other words,
for these lungs the probability that at least five of the eight
parameters were not different from normal was <1%.
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Fig. 8.
z Scores for parameters estimated from
[14C]diazepam,
[3H]alfentanil,
[14C]lidocaine, and
[3H]codeine data for
isolated lungs vs. time after CFA treatment. Dashed lines,
z = ±3.
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| |
DISCUSSION |
The objective of this study was to determine whether a change in lung
tissue composition resulting from an inflammatory response would have
differential effects on the extraction patterns for a group of
lipophilic amine compounds having different physical-chemical properties. The affirmative result is a necessary condition in support
of the hypothesis that the extraction patterns, expressed quantitatively in terms of the MID model parameter vector (a point in
N-dimensional space, where
N is the number of parameters), can
distinguish among lung phenotypes. To put this concept in perspective,
in the past the MID method has been applied to the lungs primarily to
measure extravascular lung water, capillary permeability, and metabolic
functions of the luminal endothelial surface (8-12, 16, 17,
19-22, 35, 36, 39). These applications have been shown to be
useful experimental tools (8-12, 16, 17, 19-22, 35, 36, 39)
with some clinical utility (11, 20, 39), and they have motivated
development of the theoretical basis for MID data analysis (1-5,
19-22, 35, 47). However, except for lung water, the MID method has
had little application for probing beyond the pulmonary endothelial
surface. With appropriate indicators, the MID method has the potential
for providing information about the lung tissue composition and
intracellular function that has not been previously exploited. However,
one problem has been that because the primary function of lung
perfusion is to serve the gas exchange requirements of the body, lung
perfusion is in tremendous excess relative to lung cell requirements
for typical substrates of intermediary metabolism. Therefore, there is
little expectation that a reference indicator-test indicator difference will be detectable for most endogenous hydrophilic substrates and
products. One approach to this problem for MID studies of the
physiological and pathophysiological status of the lungs has been to
use substrates for certain metabolic functions that occur on the
endothelial surface (10, 11, 16, 35, 36). Similar to the gas exchange
functions of the lungs, these functions are apparently directed at
controlling arterial blood composition rather than lung cell function,
and, therefore, to be effective, they occur at rates consistent with
the high rate of pulmonary blood flow. This has made MID measurement of
their reaction kinetics feasible (10, 11, 16, 35, 36). Measurement of
these functions can be useful if the endothelium is a focus of a
particular lung injury (10, 11, 16, 35, 36), although even then these
functions may not be tightly coupled to those intracellular functions
of the endothelial cell that are of key importance to the viability of
the endothelial cells themselves (11). Thus the probes that have been
used have had limited access to the many aspects of lung cell function
and tissue composition that may be altered by lung injury and disease.
To take greater advantage of the MID potential, the present approach
attempts to exploit a key strategy of pharmacotherapeutics, wherein a
large fraction of therapeutic drugs, as well as agents used to
manipulate cell function experimentally, come from the chemical class
of lipophilic amine compounds (23). The reasons for this include the
fact that compounds in this class have a high propensity for
associations with biomacromolecules and intracellular organelles (1,
10, 14, 15, 26, 28-32, 37, 41, 42-48). Sometimes these
interactions mimic or antagonize the action of some endogenous ligand,
usually of an ostensibly very different chemical form. Another reason
for the predominance of lipophilic amines in pharmacology is that, in
contradistinction to many endogenous substrates and ligands that do not
readily pass through capillary or cell membranes, lipophilic amines
have relatively free access to extravascular and intracellular binding
sites (1, 3, 12, 47). Thus these sites of action can be manipulated
from the bloodstream by use of lipophilic amines. However,
cell-permeant compounds with affinities for various macromolecules and
organelles are not limited to those of known pharmacological
application, and their propensity for a broad range of
biomacromolecular affinities is also the basis for their use as
molecular probes in in vitro cell biology (23).
The present study is based on the concept that one might take advantage
of the differential binding of lipophilic amines to macromolecules and
partitioning among subcellular organelles to distinguish among lung
tissue phenotypes. There is no requirement that the test indicators
have known macromolecular association sites as long as a differential
pattern of associations can be detected and correlated with some other
measure of the organ status. On the other hand, it is presumed that
identification of association sites will result in increased
specificity, and identification of such associations could be important
for understanding mechanisms of disease as well. In the context of the
present study, the large increases in the diazepam and lidocaine
sequestration per unit 3HOH
accessible extravascular lung water volume shown in Table 4 might
suggest investigation into the possibility that this inflammatory
response involves increased peripheral or mitochondrial benzodiazepine
receptors for which diazepam and lidocaine have an affinity (33, 46).
An interesting feature of the CFA-induced inflammatory response is that
the lungs almost completely recover by ~24 wk after the initial
injury without residual fibrosis (7). The latter may suggest the
relative importance of apoptosis vs. necrosis in the
resolution of the inflammatory reaction. The caspase-3 measurements
indicate that apoptosis was, in fact, a prominent feature of this
inflammatory response (Fig. 2). Given the role of peripheral
benzodiazepine receptors in the induction of apoptosis (24), this may
provide additional motivation for evaluating the contribution of
peripheral benzodiazepine receptors to the changes in the pulmonary
disposition of diazepam and lidocaine in inflamed lungs (46).
The extent of the pulmonary uptake of a lipophilic amine compound is
determined by its physical-chemical properties, which affect its degree
of ionization, lipophilicity, and affinity for plasma proteins and
tissue macromolecules (14, 29, 44, 45). For the compounds used in this
study, the importance of affinity to plasma protein in determining
pulmonary uptake was demonstrated previously (1,3). It is reiterated by
the results of the present study, wherein the uptake of the more
lipophilic but highly plasma protein-bound alfentanil was less than the
uptake of the less lipophilic but less plasma protein-bound codeine.
The extensive first-pass uptake of chemically and pharmacologically
diverse lipophilic amine drugs by the lungs has been documented in
studies primarily directed at understanding the role of the lungs in
the pharmacokinetics of these drugs (14, 15, 27-32, 44, 45, 47).
These studies have led to some generalizations about the processes
involved in the lung uptake of lipophilic amines. This uptake is
apparently via simple diffusion of the free, nonionized form of the
amine from the plasma into the lung tissue followed by association with
intracellular macromolecules and partitioning among membranes and
within subcellular organelles (1, 31, 32, 44, 47). These associations
can be very rapidly equilibrating (relative to capillary transit time),
such that tissue diffusion and binding equilibration occur virtually instantaneously between contiguous vascular and accessible
extravascular spaces at each point along the length of a perfused
capillary (1, 3, 12, 47). Other associations are slowly equilibrating with respect to the capillary transit time (1, 15, 29, 31, 32,
44-48). These generalizations are the basis of the kinetic model
for the pulmonary disposition of lipophilic amines used to parameterize
the MID data in this study (1, 47).
Lipophilic amines have been used previously as MID test indicators to
probe intact lung function. Those studies have not pursued the concept
in depth, but they are consistent with the hypothesis that the
pulmonary uptake of lipophilic amines is dependent on the condition of
the lungs. For example, Jorfeldt et al. (27) reported a decrease in
pulmonary extraction of lidocaine in patients with "pulmonary
insufficiency" compared with a healthy group. Dargent et al. (10)
and Morel et al. (39) found that the pulmonary extraction of
radiolabeled propranolol was decreased after coronary bypass and in
patients at risk for developing acute respiratory distress syndrome.
Decreased pulmonary propranolol uptake has also been observed in human
pulmonary emphysema (41). The increased iodobenzyl-propanediamine
observed in the lungs of smokers has been attributed to elevation of
alveolar macrophages (43). Experimental studies have demonstrated how
the use of indicators with a range of physical-chemical properties can
increase the information content of the MID data. For example, Merker
and Gillis (36) used propranolol to separate the effects of changing
surface area and endothelial cell metabolism on endothelial serotonin
uptake in injured lungs. Harris et al. (21) demonstrated how the ratio
of PS values for hydrophilic and
amphipathic indicators could be used to distinguish changes in
capillary permeability from changes in perfused surface area. It has
been suggested that measurement of the uptake of labeled lipophilic
amines by nuclear medicine residue detection methods may have
diagnostic utility (42). The inflammation-induced changes in lung
persistence of the amines used in the present study are consistent with
that suggestion.
Previously, we found that the parameter vector obtained with a group of
indicators
([14C]diazepam,
3HOH, and
[14C]PEA, along with a
vascular reference indicator) having different physical-chemical
properties could provide a signature with specificity for several
experimentally induced variations in lung tissue composition (47). The
present study extends those observations by demonstrating the
differential change in extraction patterns for a group of lipophilic
amines in a model of pulmonary inflammation. It seems clear that the
pattern changes reflect differential changes in the tissue sites of
association of these compounds. A question to be addressed in future
research is to what extent the pattern changes might distinguish among
disease models resulting in qualitatively and/or quantitatively
different lung cellular and macromolecular compositions.
The MID method will of course only detect the presence of sites of
associations to which the test indicator has access via the flowing
perfusate. That access can be affected by alterations in the capillary
perfusion, and part of the increased tissue mass that occurred in this
model of inflammation was not accessible to
3HOH; i.e., the fraction of lung
water detected [QW/(lung wet dry weight)] was lower in the inflamed lungs than in the
normal lungs and, therefore, presumably inaccessible to the other test indicators as well. The MID parameters
QR,
QS, and
kseq are
extensive parameters (similar to mass and heat) reflecting the amount
of accessible sites of association. However, it is also useful to know
whether there is a change in the relative amounts per unit of
accessible tissue. Ratios of these extensive parameters obtained for
different indicators having extraction patterns differentially affected
by the changes in lung composition are intensive parameters (similar to
concentration and temperature) that reflect the relative amounts of the
perfused tissue as exemplified by the normalization to the
3HOH-accessible extravascular lung
water volume (QW) in Table 4. Thus these ratios reflect the changes in perfused tissue composition independently of changes in the amount of accessible tissue.
In conclusion, this study demonstrates that the pulmonary disposition
of these lipophilic amine indicators depends on the composition of the
lung tissue. The results are encouraging with respect to the potential
use of this or another combination of lipophilic amine compounds as
indicators in the MID method for detecting and quantifying changes in
lung tissue properties associated with lung disease or injury.
| |
ACKNOWLEDGEMENTS |
This study was supported by the Whitaker Foundation, the Department
of Veterans Affairs, the Falk Trust, and National Heart, Lung, and
Blood Institute Grant HL-24349.
| |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. H. Audi,
Research Service 151, Zablocki VA Medical Center, 5000 W. National
Ave., Milwaukee, WI 53295-1000 (E-mail:
audis{at}vms.csd.mu.edu).
Received 22 February 1999; accepted in final form 8 July 1999.
| |
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TOP
ABSTRACT
INTRODUCTION
