Carbon monoxide disposition in the perfused rat liver
David G.
le Couteur1,2,3,
Zhan
Li
Yin1,
Laurent P.
Rivory2, and
Allan J.
McLean1,3,4
1 Canberra Clinical School of
the Sydney University, The Canberra Hospital, Garran, Australian
Capital Territory 2605;
2 Department of Pharmacology and
3 Department of Medicine, The
University of Sydney, Sydney, New South Wales 2006; and
4 John Curtin School of Medical
Research, Australian National University, Canberra, Australian Capital
Territory 2602, Australia
 |
ABSTRACT |
A simple method for determining carbon monoxide
(CO) disposition in the rat liver perfused with erythrocyte-free buffer
was developed. Wash-in experiments were performed with buffer
containing tracer quantities of
[14C]sucrose and
3H2O
and equilibrated with CO. Outflow samples were collected into tubes
containing human erythrocytes, which avidly bind CO. Outflow curves
were analyzed using compartmental models. Fractional recovery of CO was
1.07 ± 0.17, and the apparent volume of distribution was 1.37 ± 0.30 ml/g of liver (n = 8). A
flow-limited model fitted the data most effectively, although estimates
of the permeability-to-surface area product were attempted using a
barrier-limited model. This technique will facilitate investigation of
the effects of disease on gaseous substrate disposition in perfused organs.
compartmental analysis; permeability-to-surface area
ratio
 |
INTRODUCTION |
THE MEASUREMENT OF THE disposition of gaseous
substrates in biological systems is technically difficult. The
multiple-indicator dilution method has been used to investigate xenon
and oxygen disposition in the perfused liver (9, 11), and
ESR spin labeling has been used to examine oxygen
transport in membranes (20, 21). In the lungs, the diffusion of carbon
monoxide (CO) across the pulmonary alveolar membrane in the lung is a
widely accepted clinical test for determining the effects of disease on
the permeability barrier for oxygen (4).
The disposition of CO in the liver is itself important. Increasingly,
it is being recognized that CO influences liver function. Endogenous CO
produced by heme oxygenase controls sinusoidal perfusion through a
relaxing mechanism that involves Ito cells (22). CO also controls
biliary function, influencing contractility via a cytochrome
P-450-dependent process (19) and bile
acid-dependent biliary transport via a cGMP-dependent mechanism (18).
Exogenous CO is a frequent cause of human mortality from accidental or
suicidal smoke inhalation. The major toxicity is caused by the binding of CO to hemoglobin, which causes tissue hypoxia. However, toxicity may
also be produced by inhibition of cytochrome
c oxidase by CO that has entered cells
(3). Finally, CO is a potentially valuable surrogate marker for the
behavior of oxygen in the liver.
We report a novel and simple technique to measure CO disposition in the
perfused liver that takes advantage of the avid binding of CO to erythrocytes.
 |
MATERIALS AND METHODS |
Animals.
Male Wistar rats (8-12 wk old, 232-452 g; from John Curtin
School of Medical Research) were maintained on standard food pellets and water ad libitum. The study was approved by the Australian National
University Animal Experimentation Ethics Committee.
Chemicals.
The following gases were obtained from Linde Gas (Canberra, Australia):
95% CO-5% CO2 and 95%
O2-5%
CO2.
[U-14C]sucrose (sp act
of 10.1 Ci/mmol) was obtained from ICN Pharmaceuticals, and
3H2O
(sp act of 100 mCi/mmol) was from Amersham Life Science
(Buckinghamshire, UK).
Liver perfusion.
Rats were anesthetized with pentobarbital sodium (60 mg/kg ip,
Boehringer Ingelheim). The abdomen was opened through a midline incision. Heparin (200 units, David Bull Laboratory) was administered via the inferior vena cava. The portal vein was cannulated with an 18G
intravenous catheter (Johnson and Johnson, Pomezia, Italy) and the
thoracic inferior vena cava with a 10-cm-length PE-240 tubing
(Critchley Electrical Products). The liver was perfused in situ with
Krebs-Henseleit bicarbonate buffer equilibrated with 95%
O2-5%
CO2 for 10-15 min to allow
the liver to stabilize. The perfusate was delivered by a peristaltic
pump (Extech Equipment) in a single-pass mode at 20-30 ml/min
measured by timed collections. The experiments were performed in a
thermostat-controlled cabinet. Viability was assessed by macroscopic
appearance, portal venous pressure measured using a vertical manometer
attached to the portal venous cannula, and oxygen consumption (AVL
automatic blood gas system, AVL Medical Instruments). Assays of outflow
samples for liver enzymes did not change after exposure to CO (alkaline
phosphatase <10 U/l, alanine transaminase <24 U/l, and aspartame
transaminase <4 U/l).
Wash-in and wash-out method.
The test perfusate consisted of Krebs-Henseleit buffer equilibrated
with 95% CO-5% CO2 and
containing tracer quantities of [14C]sucrose and
3H2O.
Sucrose is a marker for the disposition of substrates that enter the
vascular and extracellular spaces within the liver (7, 8). Water is a
marker for the disposition of substrates that enter the vascular,
extracellular, and intracellular spaces of the liver in a flow-limited
fashion (7, 8).
The inflow was changed from control perfusate to the test perfusate by
switching a custom-designed four-way valve attached to the portal
venous cannula. This valve allowed standardization of perfusion
conditions between the two circuits and separation of the control and
test perfusates when the switch was made (Fig. 1). Outflow samples were collected at 1.4-s
intervals with a modified fraction collector (Extech Equipment)
containing Eppendorf tubes that had been preloaded with 100 µl of
human erythrocytes (Red Cross Blood Bank, ACT Branch, Canberra,
Australia). Samples were collected over 5 min. Outflow samples were
weighed and placed on ice. Carboxyhemoglobin concentrations and
hematocrit were measured using a blood gas analyzer (model 865, Chiron
Diagnostics). 14C and
3H activities were measured with a
scintillation counter after addition of a scintillant cocktail. Wash-in
experiments were performed at 35°C and 25°C.
During wash-out experiments, livers were perfused for 5 min with the
test perfusate containing CO,
[14C]sucrose, and
3H2O to allow equilibration of these indicators
within the liver. Then the perfusate was switched to normal
Krebs-Henseleit buffer, and the outflow samples were collected and
measured according to the method used for wash-in experiments.
Data analysis.
The concentrations of the indicators in the outflow samples were
corrected for dilution by the erythrocytes that were used to preload
the collection tubes. The carboxyhemoglobin levels also were corrected
for the measured hematocrit. The outflow activity for each indicator
was expressed as a fraction of the measured inflow activity.
Initially, analysis of the outflow curves was attempted using a
modification of the dispersion model (10, 25). The dispersion equation,
with mixed boundary conditions, was fitted to the data in the Laplace
domain using MFILT 3.2 with a Fortran 77 compiler. The equation was the
standard equation divided by s to allow for the step
input. However, we found that the sucrose data could not be fitted
adequately with this model (data not shown), suggesting that there is a
difference in the pattern of outflow curves after bolus and step input experiments.
Instead, we found a monocompartmental model to be adequate and this was
used to analyze the sucrose profiles according to the relationship
where
Ct is the outflow concentration at
time t and Q is the flow rate. The
fitted variables were
Cmax, equivalent
to the fractional recovery of sucrose;
to, the common
transit time through catheter and nonexchanging vessels; and V, the
apparent volume of distribution of sucrose.
With flow-limited distribution, the vascular and tissue compartments
effectively become lumped and the behavior of water and CO was modeled
according to the equation
where
to and Q/V were
obtained from the corresponding sucrose outflow profile,
is the
ratio of the apparent volume of distribution of sucrose to the apparent
intracellular volume of water or CO, and
Cmax is
equivalent to the fractional recovery of water or CO.
The CO data were also analyzed using a two-compartmental
barrier-limited model (Fig. 2) according to
the equation
where
and
In this case,
to and Q/V were
also obtained from the corresponding sucrose outflow profile;
k1 and
k2 are the rate
constants for the transmembrane cellular influx and efflux of CO,
respectively.

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Fig. 2.
Two-compartmental model for a noneliminated substance used for analysis
of carbon monoxide (CO). Ci,
inflow concentration; Co, outflow
concentration; Vv, vascular volume
and extracellular volume; Vt,
tissue volume;
k1, rate constant
for influx; k2,
rate constant for efflux; Q, flow rate of perfusate.
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The permeability-to-surface area (PS) product for influx is equal to
the product of k1
and the extracellular volume, which is equivalent to the apparent
sucrose volume. The PS product for efflux is equal to the product of
k2 and the
intracellular volume. The latter is equivalent to the apparent water
volume minus the apparent sucrose volume. Mirror image equations were
used for analyses of the wash-out curves.
Statistical analysis.
Data are expressed as means ± SD. Comparisons between groups were
performed using the Student's t-test
and considered significant when P < 0.05. Sigmaplot (version 4.0, SPSS, Chicago, IL) was used for the
curve-fitting procedures with no weighting. Sigmastat (version 2.0) was
used for statistical analysis. Goodness of fit was analyzed using the
Aikaike information criteria (AIC)
where
ss is the sum of squares of the fit,
n is the number of
observations, and P is the number
of unknown parameters (1).
 |
RESULTS |
Outflow curves.
An outflow curve for a wash-in experiment is shown in Fig.
3. Sucrose quickly reaches its maximum
within 30 s. Water reaches maximum activity more slowly than sucrose,
which is consistent with the larger volume of distribution of water.
The CO profile is delayed compared with water but has a similar shape.
The scatter in the data points may be secondary to the uneven effects
of the red blood cells and loss of CO from some samples. Experiments performed in the absence of a liver to determine catheter effects revealed superposition of the three curves, all of which reached maximum within a few seconds (n = 3;
Fig. 4).

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Fig. 3.
Outflow curves for sucrose, water, and CO for a wash-in experiment.
Solid lines are the fitted values for the flow-limited models for
sucrose and water. Dotted line shows the fitted values for the
barrier-limited model for CO.
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Fig. 4.
Catheter experiments showing superposition of the outflow curves for
sucrose, water, and CO. Results are means ± SD;
n = 3.
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Recovery and volume of distribution.
The recoveries and volumes of distribution of sucrose, CO, and water
are shown in Table 1. The recoveries of
indicators appeared complete. The volume of distribution of CO was
greater than that of water at 35°C
(P < 0.01), and, at 25°C, the
volume of distribution of CO was nearly double the volume of
distribution of water (P < 0.05).
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Table 1.
Volumes of distribution and recoveries of sucrose, water, and carbon
monoxide in wash-in and wash-out experiments
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Compartmental analyses.
The compartmental model appeared to fit the data adequately (Fig. 3).
The r values for the curve-fitting
procedures were 0.97 ± 0.01 for sucrose, 0.96 ± 0.03 for water,
and 0.97 ± 0.01 for CO using the flow-limited model and 0.94 ± 0.03 using the barrier-limited model. The AIC value for CO was better
using the flow-limited model (
35 ± 8) than using the
barrier-limited model (
18 ± 5). The values for the rate
constants and PS products for CO determined by this modeling are shown
in Table 2. In one experiment at 25°C, the curve-fitting program was unable to return an estimate of the rate
constants.
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DISCUSSION |
Here, we describe a simple and novel technique for measuring the volume
of distribution and barrier to cellular uptake of CO in the liver. It
has a number of advantages. Hemoglobin has a dramatic effect on the
disposition in the liver of gases such as oxygen (11) and xenon (9). In
our case, the absence of erythrocytes in the perfusate means that the
data were not contaminated by the effects of hemoglobin binding. CO is
not metabolized in the liver to any extent; therefore, we did not need
to modify our analysis to allow for sequestration or metabolism. These
characteristics mean that any differences between the outflow curves of
sucrose and CO are caused solely by the volume of distribution of CO
and any barrier to the cellular uptake of CO.
We used tubes preloaded with erythrocytes for the collection of the
outflow samples. CO avidly binds hemoglobin with a half-life at room
temperature of ~6 h. The erythrocytes effectively trap CO in the
outflow samples, overcoming the need for complex anaerobic collection
devices. Other investigators have used techniques such as a fraction
collector submerged in mercury (11) and an automated device connected
to syringes (14) to achieve anaerobic gas collection. In addition,
binding of CO to hemoglobin simplifies the measurement of CO
concentrations, which was performed using a blood gas machine with the
capacity to detect carboxyhemoglobin. Wash-in experiments use unlabeled
CO, which is technically much simpler than multiple indicator dilution
experiments that require radiolabeled gases.
The physiological behavior of other substrates in the liver has been
extensively investigated in the liver using multiple indicator dilution
experiments. In these experiments, a bolus of indicators is injected
into the inflow. Models that take into account transit time
heterogeneity (7-10, 25) are used to analyze the outflow data. We
were unable to fit our data to this type of model and found that simple
compartmental analyses appeared to describe the data adequately.
However, the problems we encountered in fitting the data did not relate
to the fitting of the diffusible tracers. Rather, the dispersion model
appeared not to be able to account for the shape of the sucrose curve.
With these compartmental analyses, the regression coefficients for all
curves were >0.9. The apparent volumes of distribution determined by
this type of model for sucrose and water are similar to those reported
with the multiple indicator dilution and analyses according to the
Goresky models (7, 8). Furthermore, the data conform to the linear
superposition principle (7). The outflow curves for water and CO are
superposed on the outflow curve for sucrose after the time points are
corrected for to
and then multiplied by the ratio of the volume of distribution of sucrose to the volume of distribution of water and CO, respectively (Fig. 5). This indicates that the
difference in the shape of the water and CO curves respective to the
sucrose curve is accounted for primarily by the larger volume of
distribution of these two markers.

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Fig. 5.
Linear superposition principle applied to the outflow curves shown in
Fig. 3. Outflow curves for water and CO are superposed on the outflow
curve for sucrose after the time points are corrected for the common
transit time through catheter and nonexchanging vessels and then
multiplied by the ratio of volume of distribution of sucrose to volume
of distribution of water and CO, respectively.
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Using this technique, we are able to report, for the first time, the
disposition of CO in the liver. We found that the apparent volume of
distribution of CO was greater than that of water at 35°C (1.37 ± 0.30 vs. 1.14 ± 0.16 ml/g of liver) and at 25°C
(1.87 ± 0.60 vs. 0.99 ± 0.22 ml/g of liver). In comparison, the
volumes of distribution of oxygen and xenon measured in the perfused
rat liver with the multiple indicator dilution method were 2.46 ml/g of
liver (11) and 1.79 ml/g of liver (9), respectively. Our results
suggest that CO partitions slightly more into liver tissue, particularly at lower temperatures. The reason for this temperature dependence is unclear. It is possible that the dissociation of CO from
liver proteins such as cytochrome c
oxidase (3) and cytochrome P-450
enzymes (23) is decreased at lower temperatures. It is also possible
that phase transitions that occur in some lipid membranes between
20°C and 37°C (6) influence membrane transport asymmetrically.
It is of note that at 25°C the apparent PS for efflux was less than
the PS for influx, which is consistent with asymmetric barrier to
membrane transfer. In contrast, the distribution of oxygen in the liver
is not influenced by temperature (11). The recovery of CO was complete,
which indicates that minimal CO is sequestered irreversibly in the
liver at physiological temperatures using this method.
The flow-limited model fitted the data more effectively than the
barrier-limited model, and the CO curves were able to be superposed
onto the sucrose curves. This indicates that CO is effectively a
flow-limited substrate and there is a minimal permeability barrier to
cellular uptake. Nevertheless, we attempted to make an estimate of this
permeability barrier using a barrier-limited model because we are
interested in applying this technique to conditions in which the
barrier to uptake could be increased. At best, these estimates of the
rate constants and PS products are a lower bound of the actual values.
The rate constants for transfer were very high, and, when values were
>2 s
1, the curve-fitting
software returned large standard errors. This characteristic has been
reported previously (11). The explanation is that barrier-limited
models become flow-limited models when the rate constants for transport
approach infinity (11). Large values cause unreliability of the
estimates for the rate constants or failure of software to fit the
data. Nevertheless, we were able to achieve an estimate for the PS
products for CO at 35°C with wash-in experiments. The PS products
were large (0.21 ± 0.11 and 0.16 ± 0.10 ml · s
1 · g
liver
1 for influx and
efflux, respectively). These values are similar to those we reported
for dimethyloxazolidinedione (0.8 ± 0.5 and 0.5 ± 0.1 ml · s
1 · g
liver
1 for influx and
efflux, respectively), which is thought to be transported across the
liver cell membrane by simple diffusion (13). As a comparison, it has
been estimated that the PS product for oxygen across cell membranes in
the heart is ~0.3
ml · s
1 · g
heart
1 (5). The value for
influx was not significantly different from the value for efflux, which
implies that the barrier to membrane transfer is symmetrical. The
surface area of hepatocytes has been estimated to be 5,600 cm2/g liver in the rat (2).
Therefore, the permeability coefficient for CO is likely to be in the
order of 4 ± 2 × 10
5 cm/s. The PS products
generated by the wash-out curves were significantly higher than those
generated by wash-in curves. This simply may reflect uncertainty in the
curve-fitting process and the fact that wash-out experiments are likely
to be unreliable for the measurement of influx. However, it is
important to note that, in the wash-out experiments, the livers were
perfused for 5 min with the test perfusate containing CO to achieve
steady-state equilibration. It is plausible that CO influences liver
cell membrane permeability indirectly through inhibition of cytochrome
c oxidase (3). Because of this latter
possibility, it appears that wash-in experiments are more suitable for
studying CO behavior than wash-out experiments.
We also performed experiments at 25°C to determine the activation
energy for the transport of CO across the cell membrane. By application
of the Arrhenius equation to the values for the rate constants at
35°C and 25°C, we found that the activation energy for the
influx of CO was ~0.6 kcal/mol. Our value is less than values
reported for oxygen diffusion in erythrocyte membranes [3
kcal/mol (6), 2.6 kcal/mol (24)], frog sartorius muscle [3.85 kcal/mol (15)], and phosphatidyl-cholesterol
membranes [3.7-6.5 kcal/mol (21)]. Overall, the
combination of symmetrical barrier to membrane transfer, high PS
products, and an activation energy lower than the value for the
diffusion of oxygen across cell membranes suggests that CO also crosses
cell membranes by simple diffusion but does not exclude a rapid carrier mechanism.
This technique proved to be a simple and effective method for
determining the behavior of CO in the perfused rat liver. It does not
require complex anaerobic collection devices, radiolabeled gases, or
complex analytical equipment; accordingly, the method should be widely
accessible. The technique may have wider applications than simply the
study of CO disposition. In clinical medicine, the diffusibility of CO
across the capillary membrane of the lung has been used extensively as
a surrogate marker for measuring the barrier-to-oxygen transfer in
pulmonary disease (4). The study of oxygen transfer in the liver is
also of potential importance in disease. We have postulated that
impaired transfer of oxygen into the liver cells causes some of the
metabolic and drug detoxification changes that have been observed in
cirrhosis of the liver and aging. In cirrhosis of the liver, we suggest
that the permeability barrier to oxygen transfer lies at the level of
the capillarized sinusoid (16). In the aging liver, we propose that the
permeability barrier occurs at the level of the cell membrane (12).
This technique will allow the investigation of gas transfer into the hepatocytes of aged and cirrhotic livers. In addition, the technique is
applicable for studying gas transfer in other diseases and other
perfused organs.
In conclusion, we have developed a simple method for measuring CO
behavior in the perfused rat liver. It is likely that CO crosses the
hepatocyte membrane by diffusion with a small but nevertheless
measurable barrier to transfer. The method may have wider experimental
use as a surrogate marker for the hepatic disposition of oxygen in disease.
 |
ACKNOWLEDGEMENTS |
We acknowledge the technical support of Lionel Davies and the
Biochemistry Department of The Canberra Hospital.
 |
FOOTNOTES |
This study was supported by the National Health and Medical Research
Council of Australia, Private Practice Trust Fund of The Canberra
Hospital, and the University of Sydney Research Grants.
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: A. J. McLean,
Dept. of Medicine, The Canberra Clinical School of the Univ. of Sydney,
The Canberra Hospital, Yamba Drive, Garran, ACT 2605 Australia (E-mail:
allan_mclean{at}dpa.act.gov.au).
Received 7 December 1998; accepted in final form 3 June 1999.
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