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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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Fig. 1.   Design of the 4-way valve used to switch between control and test perfusates.

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
C<SUB>t</SUB> = C<SUB>max</SUB>(1 − <IT>e</IT><SUP>−Q(<IT>t − t</IT><SUB>o</SUB>)/V</SUP>)
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
C<SUB>t</SUB> = C<SUB>max</SUB>(1 − <IT>e</IT><SUP>−Q(<IT>t</IT> − <IT>t</IT><SUB>o</SUB>)/V(1 + &thgr;)</SUP>)
where to and Q/V were obtained from the corresponding sucrose outflow profile, theta  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

C<SUB>t</SUB> = C<SUB>max </SUB><FENCE>1 + <FR><NU>C<SUB>max</SUB>Q</NU><DE>V(<IT>b</IT> − <IT>a</IT>)</DE></FR> <FENCE><IT>k</IT><SUB>2</SUB> <FENCE><FR><NU><IT>e</IT><SUP>−<IT>b</IT>(<IT>t</IT> − <IT>t</IT><SUB>o</SUB>)</SUP></NU><DE><IT>b</IT></DE></FR> − <FR><NU><IT>e</IT><SUP>−<IT>a</IT>(<IT>t</IT> −<IT>t</IT><SUB>o</SUB>)</SUP></NU><DE><IT>a</IT></DE></FR></FENCE> + <IT>e</IT><SUP>−<IT>a</IT>(<IT>t</IT> − <IT>t</IT><SUB>o</SUB>)</SUP> − <IT>e</IT><SUP>−<IT>b</IT>(<IT>t</IT> − <IT>t</IT><SUB>o</SUB>)</SUP></FENCE> </FENCE>

where
<IT>a</IT> = <FENCE>(<IT>k</IT><SUB>1</SUB> + <IT>k</IT><SUB>2</SUB> + Q/V) − <RAD><RCD>(<IT>k</IT><SUB>1</SUB> + <IT>k</IT><SUB>2</SUB> + Q/V)<SUP>2</SUP> − 4<IT>k</IT><SUB>2</SUB>Q/V</RCD></RAD></FENCE><FENCE>2</FENCE>
and
<IT>b</IT> = <FENCE>(<IT>k</IT><SUB>1</SUB> + <IT>k</IT><SUB>2</SUB> + Q/V) + <RAD><RCD>(<IT>k</IT><SUB>1</SUB> + <IT>k</IT><SUB>2</SUB> + Q/V)<SUP>2</SUP> − 4<IT>k</IT><SUB>2</SUB>Q/V</RCD></RAD></FENCE><FENCE>2</FENCE>
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.

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)
AIC = <IT>n</IT> log (<IT>ss</IT>) + 2<IT>P</IT>
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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

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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Table 2.   Rate constants and PS products for the influx and efflux of carbon monoxide


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 277(3):G725-G730
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