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1 Biomedical Engineering
Department, 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
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).
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
EXPERIMENTAL RESULTS
DATA ANALYSIS
MODEL RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
EXPERIMENTAL RESULTS
DATA ANALYSIS
MODEL RESULTS
DISCUSSION
REFERENCES
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 |
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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) |
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EXPERIMENTAL METHODS |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
EXPERIMENTAL RESULTS
DATA ANALYSIS
MODEL RESULTS
DISCUSSION
REFERENCES
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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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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.| |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
EXPERIMENTAL RESULTS
DATA ANALYSIS
MODEL RESULTS
DISCUSSION
REFERENCES
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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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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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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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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)
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ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
EXPERIMENTAL RESULTS
DATA ANALYSIS
MODEL RESULTS
DISCUSSION
REFERENCES
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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
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(2) |
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(3) |
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
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(4) |
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.
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MODEL RESULTS |
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ABSTRACT
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DATA ANALYSIS
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REFERENCES
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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.
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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.
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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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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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
EXPERIMENTAL RESULTS
DATA ANALYSIS
MODEL RESULTS
DISCUSSION
REFERENCES
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
EXPERIMENTAL RESULTS
DATA ANALYSIS
MODEL RESULTS
DISCUSSION
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) 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 (