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Vol. 30, Issue 3, 307-313, March 2002
Laboratory of Exposure Assessment and Environmental Epidemiology (B.H., C.R., K.v.T., M.H., J.v.E.) and Laboratory for Pathology and Immunobiology (J.D.), National Institute of Public Health and the Environment, Bilthoven, The Netherlands
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Liver slice experiments were performed to determine the slice
intrinsic clearance and to extrapolate this to the in vivo liver intrinsic clearance in a physiologically based pharmacokinetic (PBPK)-like approach. Precision-cut liver slices were incubated with
different initial concentrations of tolbutamide, and the time series of
parent and metabolite concentrations were measured in slice and
incubation medium. A mathematical model was built that modeled the
uptake of tolbutamide and its metabolism in the liver slice. In
addition, binding of tolbutamide to cellular constituents and partition
over the water and lipid phase were accounted for by the model. Model
analysis imposed sampling of parent compound in slice and of
metabolites pooled from slice and medium. The model was calibrated to
the data, fitting the intrinsic clearance, the parent compounds' free
fraction in liver material, and a diffusion parameter describing
medium-slice exchange of tolbutamide. In addition, to ensure a
meaningful application of the theoretical model, slice viability
parameters were monitored before and during the experiment. For the
different incubations, the intrinsic clearance per unit of volume of
slice ranged from 0.035 to 0.086 min
1 when not correcting
for slice viability and from 0.044 to 0.11 min1 when
correcting for slice viability. The results were extrapolated to a PBPK
model for tolbutamide in the rat. The value for the intrinsic
clearance found by calibrating the PBPK model to previous in vivo data
was 0.090 min1. This result suggests that liver slices
are a valuable tool for predicting in vivo intrinsic clearance of
low-extraction compounds.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the past decade, precision-cut liver slices
have been successfully used as an in vitro model for biotransformation
(Dogterom and Rothuizen, 1993
; Ball et al., 1996; Hashemi et al.,
1999), hepatotoxicity (Wormser et al., 1990; Miller et al., 1993), and enzyme induction studies (Lake et al., 1997) in a variety of animal species. Important determinants in this successful application of
liver slices are undoubtedly the maintenance of normal tissue architecture, cell heterogeneity, and cell-cell communications within
the livers' original tissue matrix.
However, a drawback of liver slices often addressed in studies
predicting rates of metabolism is their diffusional limitations (Ekins
et al., 1995; Worboys et al., 1997). The outer cell layers of liver
slices are directly exposed to incubation media; however, the inner
cell layers are only exposed to the test compound when it has traveled
through or around the outer cell layers. This phenomenon has been
visualized using a fluorescent dye (Ekins et al., 1995). Diffusional
limitations have often led to underevaluation of in vivo intrinsic
clearance rates when liver slice data are normalized for
hepatocellularity (Worboys et al., 1996a,b).
These problems, however, can be counteracted when appropriate modeling
of in vitro data is incorporated in the prediction strategy. In this
respect, it is important to note that Worboys et al. (1997) assessed
drug metabolism in rat liver slices by analyzing experimental data with
a classical one-compartment model. However, the liver slice model
consists of two physically different phases (i.e., slice and
surrounding incubation medium), and hence, a two-compartment model
seems more appropriate. In contrast to other liver slice models
designed to predict rates of metabolism we developed a model that
consists of a physicochemical part, a mathematical part, and an
observable part. Our liver slice model takes into consideration
processes of transport, partitioning, and elimination of drug and/or
its metabolites, which leads to the identification of the ultimate
parameter: the slice metabolic rate constant
CLs.1
The development of our rat liver slice model can be seen as a first step to improve the applicability of PBPK models for human risk analysis.
We present here the experimental validation of our model approach by
using tolbutamide as model compound that is metabolized by rat liver
slices into hydroxytolbutamide and carboxytolbutamide. To test the
validity of our model approach, we extrapolated our in vitro data to an
existing PBPK model for tolbutamide in the rat (Sugita et al., 1982).
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals.
Tolbutamide and chloropropamide were obtained from Sigma Chemical Co.
(Zwijndrecht, The Netherlands). Hydroxytolbutamide and carboxytolbutamide were purchased from Brunschwig (Amsterdam, The
Netherlands). Testosterone (4-androsten-17
-ol-3-one),
4-androsten-11,17-diol-3-one, 4-androsten-2
, 4-androsten-6,
4-androsten-7, 4-androsten-16, 4-androsten-16, and
4-androstene-3,17-dione were obtained from Steraloids (Wilton,
NH). Williams' medium E (WME) supplemented with Glutamax I was bought
from Invitrogen (Paisley, Scotland). All other chemicals and
solvents were of analytical and HPLC grade, respectively.
Animals. Adult male Wistar (HsdCpb:WU) rats (230-300 g) were obtained from Harlan CPB (Zeist, The Netherlands). Animals were allowed free access to water and food (Hope Farms, Woerden, The Netherlands).
Slice Preparation. The preparation of liver slices was performed according to a standard operating procedure. Animals were anesthetized by asphyxiation by means of CO2/O2, and the livers were excised and weighed. The liver was placed on a silicon support and liver cores (approximately 9 mm in diameter) were made by means of a drilling machine. Precision-cut liver slices were prepared in ice-cold WME saturated with 95% O2, 5% CO2 (carbogen) by using a Krumdieck tissue slicer (Munford, AL). Slice thickness was checked intermittently by eye. The slicer was disassembled and cleaned between experiments.
Slice Incubation. Only intact slices, round or oval, were used. Three liver slices (each slice from a different rat liver) were put on a stainless steel insert (Vitron, Tucson, AZ), placed in a scintillation vial containing 2.8 ml (optimal volume for these vials) of prewarmed (37°C) and pregassed (carbogen) WME. Regarding the intended incubation time (a few hours, maximally), slices were not continuously gassed with carbogen. Instead, before transfer of the vial into the roller incubator the void space of the vial was saturated with carbogen. Vials were rotated in a prewarmed (37°C) roller incubator at 2 rpm.
After incubation, the vials were transferred to ice. Slices (except those for histomorphology) were removed from the wire mesh, gently dried on filter paper, and transferred to 2.8 ml of ice-cold WME. Slices were disrupted by sonification for 15 min (Branson sonifier 250; Branson, Danbury, CT) on crushed ice.Liver Slice Model.
A detailed description of the liver slice model is depicted in van
Eijkeren (2002). The main characteristics are briefly summarized. The
liver slice model consists of three models: a physicochemical model
(describing the slice experiment), a (mathematical) system model, and
an observable model (Fig. 1). The model
was built together with a set of eight model parameters: the initial
concentration (Cm,0) of the parent
compound; medium volume (Vm) and slice
volume (Vs); the diffusion coefficient
for parent drug (D); the free fractions in incubation medium
and slice for parent compound (fm, fs), respectively; the
octanol-water-based liver slice/culture medium partition
(P
1, 2, and 
) is
depicted in Fig. 1, bottom right. It shows observations of parent
compound in slice from a simulated experiment (*), from which the
initial (1) and terminal
(2) phase rates and concentration scaling
() can be identified [e.g., by fitting the analytical solution
(straight line)]. These observable parameters derive from the system
parameters. The liver slice intrinsic clearance of tolbutamide
was calculated by means of a mathematical model with the ACSL Math
package (www.inpol.com/acsl), as described by J. van Eijkeren
(2002).
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Kinetic Profiles for Tolbutamide Metabolism.
Three slices per vial were incubated with 40, 90, 125, and 170 µM
tolbutamide (CP,m,0) in 2.8 ml of WME
(Vm). These concentrations are actual
concentrations and were approximately 85% of the intended concentrations of 50, 100, 150, and 200 µM, respectively. The amount
of tolbutamide, including metabolized tolbutamide, remained constant
during the incubation (Fig. 2). Because
the loss of tolbutamide is constant in all stock solutions, glass
adherence of tolbutamide is a likely explanation for this phenomenon.
Because the highest concentration was approximately 30% of the
Michaelis constant calculated by Worboys et al. (1995), metabolism was
assumed to be linear. Samples of slice and medium were taken separately
at 0, 2, 4, 6, 8, 10, 20, 30, 40, 50, and 60 min after incubation and
stored at 
20°C. Samples were analyzed for parent compound (tolbutamide) and metabolites (hydroxy- and carboxytolbutamide) by
means of a validated, slightly modified HPLC method according to Back
et al. (1984). Two-milliliter aliquots of incubation medium or liver
slice homogenate were extracted with a mixture of diethyl ether/dichloromethane/iso-propanol in a 60:40:1 ratio (v/v). After evaporation of the organic layer, the residue is dissolved in eluents
(acetonitrile and
H2O/H3PO4,
pH 2.7; 1:2.4). Tolbutamide and its metabolites were separated on a
Gilson 307 HPLC by an isocratic method.
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Slice Characterization.
Determination of
Vs
To this end, the slice wet weight was determined. Fifteen
representative slices of each rat liver were selected by eye and transferred to prewarmed (37°C) WME and incubated for 10 min at 37°C. After this, the slices were dried on filter paper and
weighed. By dividing the slice wet weight by 1.04 g/cm3 (i.e., the specific gravity of human liver)
(ICRP, 1992), the liver slice volume
(Vs) was calculated. By doing so, it was
assumed that the specific gravity of human and rat liver is similar.
Slice Viability. The viability of tolbutamide-treated slices was assessed histologically by evaluating the eosinophilic staining of the cytoplasm as well as the detached location of cells. Slices were incubated for different lengths of time with tolbutamide (40-170 µM). After incubation, slices were fixed in 4% phosphate-buffered formaldehyde. After dehydration and embedding of liver slices in paraplast, 5-µm sections were made and stained with hematoxylin-eosin. Slice thickness in cross sections was determined at a minimum of five sites of the slice by means of an interactive image analysis system (IBAS 2000; Kontron, Munich, Germany). In addition, in these sections the number of total cell layers and nonvital cell layers was determined.
Determination of fP,m.
Although this parameter was set to 1 due to the omission of protein in
the incubation medium, this assumption was validated by characterizing
both slices and incubation medium for protein content (Lowry et al.,
1951). Because tolbutamide is an acidic drug that is highly bound
primarily to the albumin fraction of plasma protein (Judis, 1972), the
albumin content of slices and incubation medium was also assessed
(Doumas et al., 1971).
Biotransformation Capacity.
To this end, liver slices were incubated for 1 h with 250 µM
testosterone after different times of preincubation (0-4 h). The
amount of metabolites formed was determined by HPLC according to van't
Klooster et al. (1993).
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Determination of Vs. The slice volume in the different tolbutamide experiments can be summarized as follows (mean ± S.D.): 13.2 ± 5.0 mm3 (40 µM TOL), 15.2 ± 3.5 mm3 (90 µM TOL), 20.1 ± 4.0 mm3 (125 µM TOL), and 19.2 ± 5.4 mm3 (170 µM TOL). The thickness of the liver slices (mean ± S.D.; n = 60) was 259 ± 54 µm and the mean thickness of one cell layer was calculated to be 15 µm.
Slice Viability. This was evaluated both as function of time and of tolbutamide concentration and the results are shown in Table 1. Tolbutamide did not affect the number of nonvital cell layers. However, as the experiment progressed, a slight increase in the number of nonvital cell layers was observed at 40 and 60 min of incubation. At these time points, a disconnected line of nonvital cells was observed on both sides of the slice, whereas at earlier time points, the nonvital cells still formed part of the slice core.
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Determination of fm. The amount of protein leakage from liver slices was maximally 3 to 4% of the total amount of cellular protein. Approximately one-half of the protein leakage was accounted for by albumin (data not shown). Considering the coefficient of variation of maximally 10% for our HPLC method, the observed protein leakage most probably had no measurable consequences for the free fractions of tolbutamide and its metabolites.
Determination of P). This algorithm requires the water and
lipid contents of rat liver tissue as a fraction of tissue weight (0.7 and 0.06, respectively; Poulin and Krishnan, 1995) and the subdivision
of lipids in phospholipids and neutral lipids (0.42 and 0.58, respectively, as fraction of total lipids; Poulin and Krishnan, 1995).
The water content of culture medium was estimated to be 1. Taking the
log (Kow) value to be the log (D)7.4 = 0.52 value for tolbutamide presented in
Worboys et al. (1997), this algorithm yields a value of
P
Biotransformation Capacity. The metabolism of testosterone by liver slices after different times of preincubation is depicted in Fig. 3. Increasing the preincubation time, a slight but consistent reduction in metabolic capacity was observed. Therefore, because reliable measurement of in vitro rates of metabolism are of pivotal importance for an adequate calculation of the slice's intrinsic clearance it was decided that incubations of liver slices with tolbutamide were carried out without prior preincubation.
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Model Fitting of Experimental Data.
In van Eijkeren (2002) it is shown that for a meaningful data analysis
of the parameters for drug metabolism, CLs,
drug-free fraction, fs, and exchange
of drug between culture medium and liver slice, D, a
simultaneous fit of the amount of tolbutamide in slice and the sum of
the amounts of the metabolites hydroxy- and carboxytolbutamide pooled
from slice and medium are required. Moreover, for checking the mass
balance, the initial amount of tolbutamide in the medium was calculated
by a simultaneous fit of the amount of tolbutamide in culture medium as
well. All calculations were performed with the ACSL-Optimize package,
optimizing the log-likelihood of the parameter values, considering the
experimental data, and assuming a relative error model. Fitted values
appear in the text with standard errors (obtained from the Hessian).
it is assumed that the
free fraction of any compound never exceeds the value 1, model fits for
the various incubations were performed while not imposing this bound.
For the 170 and 125 µM incubations values of 1.04 (0.07) and 1.09 (0.06), respectively, were fitted for this parameter. This is a slight
overestimation, but the value 1 lies within the 95% range of the
values found. Also, because for the 90 and 40 µM incubations values
of 0.79 and 0.64, respectively, were found, in the following text all
references to fitting data assume an upper bound of 1 be set for the
free fraction of tolbutamide in liver slice. The values found for the
free fraction of tolbutamide in slice are 0.64 (0.08), 0.79 (0.11), 1 (0.13), and 1 (0.07) for the 40, 90, 125, and 170 µM incubations, respectively.
In van Eijkeren (2002), the metabolism parameter CL describes the
intrinsic liver slice clearance for the whole slice. For extrapolation
purposes, we are interested in the specific intrinsic clearance (i.e.,
the intrinsic clearance per volume of liver slice). The fitted values
of this parameter are 0.086 (0.020) min1 for
the 40 µM incubation, 0.059 (0.013) min1 for
the 90 µM incubation, 0.035 (0.006) min1 for
the 125 µM incubation, and 0.055 (0.006) min1
for the 170 µM incubation. These values were fitted assuming the
nonviable cells to have equal metabolizing potency as the viable ones.
However, if one assumes that chemical conditions, especially cell pH,
prohibit action of the metabolizing enzymes, one should not divide the
intrinsic clearance by total cell volume, but by viable cell volume.
This procedure is justified by the assumption on well stirredness of
the liver slice. The experimental setup leads to identification of the
intrinsic clearance as a property of the liver slices as a whole:
whether the sites of clearance reside in only part of the slice or the
entire slice cannot be determined. Given the observed clearance rate
and the drug to be equally distributed within the slice (well
stirredness assumption), specific metabolism of the clearance sites
(i.e., the clearance rate per unit of volume of these sites) follows by
dividing the observed slice clearance by the clearance sites volume,
instead of by total slice volume.
From the histological results it appears that viable cell volume is
about 80% of total volume for the different incubations. This way, one
concludes to the values for the specific intrinsic liver clearance of
0.11, 0.073, 0.044, and 0.069 min1 for the 40, 90, 125, and 170 µM incubations, respectively.
The time course of the amounts for the different incubations, together
with the data, are shown for tolbutamide in incubation medium, in slice
and pooled metabolites in Figs. 2, 4,
and 5, respectively. Figure
6 shows typical behavior of the
standardized residuals with respect to the fitted time courses.
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In Vitro-In Vivo Extrapolation.
Sugita et al. (1982) present a PBPK model for tolbutamide in the rat.
In this model, metabolism is modeled with saturable Michaelis-Menten
kinetics, including a submodel for the free fraction of tolbutamide in
plasma. Their model has been implemented in ACSL, replacing
Michaelis-Menten kinetics with linear kinetics. Intrinsic liver
clearance was modeled by multiplying the value for the specific
intrinsic clearance found from the incubation experiments by liver
volume. The free fraction in blood was assumed to consist totally of
the free fraction in plasma, thus considering the fraction in
erythrocytes to be bound.
and a best fit, fitting the PBPK model specific
intrinsic clearance to these data. The value of the specific intrinsic
clearance found by their fit was 0.091 min1.
Figure 8 shows the same, but with values
for the specific intrinsic clearance obtained by dividing the liver
slice intrinsic clearances from the incubation experiments by viable
cell volume only.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Slice Characteristics. For accurate modeling of in vitro metabolism data slice viability and hence metabolic capacity need to be accurately determined. It was shown that during the relatively short incubation period (60 min), a slight increase in number of nonvital cell layers was observed. In addition, from slice histology data it was calculated that the number of viable cells amounted to 80% of total number of liver cells for the different incubations. The question rises whether nonviable cells are capable of metabolizing tolbutamide and to what extent this reaction is likely to occur in damaged tissue. Because this is of pivotal importance for accurate modeling of in vitro metabolism data we modeled our metabolism data with and without correction for the number of viable cells. Notice that this correction was effected only by dividing liver slice intrinsic clearance (found by fitting to the data) by the volume of viable cells and not by total slice volume. So doing, it was tacitly assumed that other physicochemical properties of the slice (transport, octanol-water-based partition and binding) were not dependent on cell viability.
From the slice characteristics it appears that after about 0.5 h, the outer slice layers with nonviable cells disconnect from the inside. So, model fits were also performed using data until t = 30 min only. However, no substantial differences were found with parameter estimations by using all the data, so these results are not reported. This corroborates the assumption mentioned above that cell viability did not influence the liver slice physicochemical properties, except for possible metabolism.Identification.
In van Eijkeren (2002), it is shown that the estimations for the
free fraction of tolbutamide and its intrinsic clearance by fitting the
data become unreliable when the latter value exceeds the estimation for
the diffusion parameter, whereas the value of their product remains
reliable. It appears that the value for the slice intrinsic clearance
never exceeds a fraction of 0.22 of the value of the diffusion
parameter. From this modeling point of view the fitted values can be
considered as reliable.
30%) and the diffusion
parameter (about twice as large). This does not hold for the 125 µM
parameter. Note that the data for the 40 µM incubation are erroneous.
It is concluded that perhaps a value of the intrinsic clearance in
between the values found for the 90 and 170 µM incubations is the
most reliable estimate.
One could question the approach of transport of the compound by one
diffusion parameter, lumping the processes of diffusion in culture
medium to the slice and, perhaps more importantly, of diffusion in the
slice to metabolizing sites. Stated another way: How well stirred is
the liver slice? Two elements in relation to well stirredness are
involved. The first element is the characteristic time scale of
intraslice diffusion of a compound in connection to the time scale of
observations: the smaller the latter, the smaller the first should be
so that the slice can be considered well stirred. The second element is
the characteristic time scale of specific intrinsic clearance in
connection to the characteristic time scale of diffusion. The first
should be large compared with the latter so that the slice can be
considered well stirred. This involves not only the diffusional process
itself but also slice dimensions: the characteristic time scale for
diffusion is the squared thickness of the slice divided by the
diffusion coefficient. Additionally, it is assumed that viability does
not influence the cell's diffusion characteristics, because viability
has primarily to do with biochemical processes and not with physical
ones. Note that the diffusion value is not only determined by the liver
material properties but also by the compound properties. The question
of well stirredness has been investigated by modeling intraslice diffusion explicitly (J. van Eijkeren, unpublished data).
Initial results indicate that the assumption on well stirredness
reasonably applies to the kinetics of tolbutamide in liver slices.
Worboys et al. (1995) report an intrinsic clearance for tolbutamide of
0.8 (0.6) µl/min for slices of 7.30 (0.60) mg of dry weight, which
amounts to about 10 (0.8) mg of wet weight. So, the specific intrinsic
clearance, when dividing by total slice volume, is about 0.08 (0.08)
min1. Our results, which lie in range of 0.035 to 0.086 min1, compare well with their
result. It may seem disappointing that the modeling approach in van
Eijkeren (2002) does not lead to substantially different results as
does the simple modeling approach in Worboys et al. (1997). As simple
models as possible should be used for understanding experimental
results. However, the more complex modeling in van Eijkeren (2002)
offers the key to the understanding why the different approaches lead
to almost the same result.
First, it appears that the diffusion process has a characteristic time
scale that is about 2.5 times as fast as the characteristic time of
clearance (e.g., 0.004 and 0.0001 µl/min for diffusion and clearance,
respectively). So, clearance is the rate-limiting process and
difference between the models is to be expected only when diffusion is
rate limiting. First, in van Eijkeren (2002) it is analyzed that when
diffusion is rate limiting, identifiability of slice intrinsic
clearance becomes unreliable. Second, in such a case, the assumption on
well stirredness of the liver slice becomes doubtful and one has to
resort to a model that includes intraslice diffusion (J. van Eijkeren,
manuscript in preparation). This suggests to compare the models
together with a model that includes intraslice diffusion with a
compound possessing high-clearance properties.
Second, except for the 40 µM incubation with its erratic results,
binding of tolbutamide does not seem to be of importance: if its free
fraction, for example, had been found to be 0.2 instead of 0.8 and
higher, surely the difference between the two model approaches would
have been more pronounced. This suggests to compare the two well
stirred models of van Eijkeren (2002) and Worboys et al. (1997) with a
compound with strong binding capacity.
Extrapolation. For the in vitro-in vivo extrapolation one needs the specific intrinsic slice clearance, i.e., the clearance per unit volume of slice. One of the difficulties has been alluded to above: estimation of slice volume should be fairly accurate. For the determination of slice volume, other slices, but from the same sliced batch, than those that are incubated are being used. The choice is by eye, trying to choose as good as possible slices of the same quality for incubating and for weighing.
Another problem is whether one should exclude the nonviable fraction of cells from the sites where metabolism takes place or not. On the one hand, one argues that in incubation experiments with microsomes even the total cell structure is damaged and the microsomal fraction extracted and incubated. Still, the enzymes responsible for primary metabolism are active. On the other hand, microsomes are incubated in fluid under chemical conditions that favor their functioning, whereas chemical conditions in nonviable cells, notably loss of cofactors, might prohibit such functioning. However this may be, when not correcting for nonviable volume the extrapolation result is satisfying if one keeps in mind that the livers from the incubation experiments are from rats of a different strain than the rats used in the experiments of Sugita et al. (1982). When
correcting for nonviable volume, the results are even better. Although
morphological deterioration of tissue might not be regarded as a direct
indicator of decreased biotransformation capability, the application of
this correction seems justified regarding the improvement in prediction
of the plasma tolbutamide profile.
Conclusion. In the present study we have shown that liver slice experiments can be useful in the determination of the specific intrinsic liver clearance. This usefulness depends on the ratio between the characteristic times for metabolism and diffusion of the chemical compound involved. The latter not only depends on the physical process of diffusion but also on slice thickness. Note that this observation does not exclude drugs that are known as "high-clearance drugs" from application of this in vitro technique: high clearance can be the result of both fast metabolism and fast diffusion with a ratio of characteristic times favorable for reliable identification. On the contrary, when low clearance of a compound is the result of a slow diffusion process and a fast metabolism process, such a "low-clearance drug" is expelled from application. In this sense, the distinction between low and high clearance perhaps has to be reconsidered.
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Acknowledgments |
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We gratefully acknowledge H. A. van Loenen for protein and albumin determinations. Gratitude is also expressed to H. J. Loendersloot for assistance with histology, to Dr. W. Maas for the testosterone assay, and to Drs. ir. L. L. de Zwart and A. J. A. M. Sips for assistance with the incubations. Drs. ir. L. L. de Zwart and Dr. ir. M. J. Zeilmaker are also acknowledged for critically reviewing the manuscript.
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Footnotes |
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Received April 10, 2001; accepted November 19, 2001.
Dr. H. E. M. G. Haenen, Laboratory of Exposure Assessment and Environmental Epidemiology, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands. E-mail: bert.haenen{at}rivm.nl
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Abbreviations |
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Abbreviations used are:
CLs, slice
metabolic rate constant;
PBPK, physiologically based pharmacokinetic;
WME, Williams' medium E;
HPLC, high-performance liquid chromatography;
TOL, tolbutamide;
Vs, slice volume;
P
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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), and 170 µM tolbutamide (*).
