Hepatic uptake of hippurate: a multiple-indicator dilution,
perfused rat liver study
Tsutomu
Yoshimura1,
Andreas J.
Schwab2,
Lei
Tao1,
Ford
Barker1, and
K. Sandy
Pang1,3
1 Faculty of Pharmacy,
University of Toronto, Toronto M5S 2S2;
3 Department of Pharmacology,
University of Toronto, Toronto, Ontario M5S 1S1; and
2 McGill University Medical
Clinic, Montreal General Hospital, and Department of Medicine,
McGill University, Montreal, Québec, Canada H3G 1A4
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ABSTRACT |
The hepatic transport of hippuric acid (HA), a
glycine-conjugated metabolite of benzoic acid that exhibits only modest
plasma albumin binding (binding association constant of 2.1 × 103
M
1), was studied in the
single-pass perfused rat liver (12 ml/min), using the multiple
indicator dilution (MID) technique. The venous recovery of
[3H]HA on portal
venous injection of a MID dose containing a mixture of a set of
noneliminated reference indicators and
[3H]HA revealed a
survival fraction of unity, corroborating the lack of disappearance of
bulk HA from plasma. When the outflow recovery was fitted to the
barrier-limited model of Goresky et al. (C. A. Goresky, G. G. Bach, and
B. E. Nadeau. J. Clin. Invest. 52:
991-1009, 1973), the derived influx
(PinS ) and
efflux (PoutS )
permeability-surface area products were found to be dependent on the
concentration of HA (1-930 µM);
PinS and
PoutS were ~3.5 times the plasma flow rate at low HA concentration, but decreased with
increasing HA concentration. All values, however, greatly exceeded the expected contribution from passive diffusion, because the
equilibrium distribution ratio of chloroform to buffer for HA was
extremely low (0.0001 at pH 7.4). The tissue equilibrium partition
coefficient
(Pin/Pout,
or ratio of influx to efflux rate constants,
k1/k
1)
was less than unity and decreased with concentration. The optimized
apparent Michaelis-Menten constant and maximal velocity were 182 ± 60 µM and 12 ± 4 nmol · s
1 · g
1,
respectively, for influx and 390 ± 190 µM and 29 ± 13 nmol · s
1 · g
1,
respectively, for efflux. In the presence of
L-lactate (20 mM), however,
PinS for the uptake
of HA (174 ± 3 µM) was reduced. Benzoic acid
(10-873 µM) was also effective in reducing hepatic uptake of HA
(5.3 ± 0.9 µM). These interactions suggest that MCT2, the monocarboxylate transporter that mediates the hepatic uptake of lactate
and other monocarboxylic acids, may be involved in HA transport.
benzoic acid; L-lactate; influx and efflux coefficients; permeability-surface area product; carrier-mediated transport; monocarboxylic acid transporter; MCT2; plasma and tissue protein binding; rat liver perfusion
 |
INTRODUCTION |
THE TRANSPORT OF ORGANIC anions across the basolateral
(sinusoidal) membrane of the liver has been extensively studied. Recent advances in molecular biology have revealed the existence in the liver
of ntcp, the Na+-dependent bile
acid (taurocholate) cotransporter (16, 17), and oatp, the multiple
organic anion transporter that mediates the transport of organic anions
(20, 21, 26, 27, 34) and cations (3). The carrier-mediated transport of
sulfobromophthalein (26, 27, 34) and its glutathione conjugate (13) and
sulfated estrone and bile acids and drug sulfate conjugates (11, 18, 33, 35) implicates a role for oatp or other as yet unknown transporters.
The transport of arylmonocarboxylic acids in the liver has not been
studied extensively (6, 31). The hepatic transport of the simple
arylcarboxylate anions aminohippurate and acetamidohippurate in rat
liver perfusion experiments was inhibited by probenecid (6). Transport
of the precursor, benzoate, into Caco-2 intestinal cells displayed a pH
dependence, suggesting the involvement of carrier proteins (38). In the
intestine, interaction was found between the transport of benzoate and
L-lactate (37). Transport of
L-lactate is ordinarily mediated
by the monocarboxylate transporter MCT1, which is present abundantly in
the intestine, erythrocytes, and cardiocytes (10, 36, 37). MCT2, which
is present in the liver, is responsible for the uptake of lactate and
pyruvate and is distinct from MCT1 (9). Whether the same substrate
specificity for MCT1 applies to MCT2 or whether MCT2 is capable of
transporting simple arylcarboxylic acids into liver cells is unknown.
In this study, we examined the hepatic uptake of hippuric acid (HA), a
simple organocarboxylic acid. HA is the glycine-conjugated metabolite
of benzoic acid found in almost all animal species, including humans
(4). It is present in herbivorous animals, and its existence is also
associated with its precursor, benzoate, a common food preservative.
The fate of hippurate has been studied in conjunction with benzoate in
the single-pass-perfused rat liver (5). Once formed, hippurate is
neither excreted nor further metabolized by the liver; only efflux to
the venous outflow occurs. Thus HA represents the simplest test
compound for the study of arylcarboxylic acids. Preliminary plasma
protein-binding experiments have demonstrated that HA is bound only to
albumin and not to red blood cells (RBC). We used the
multiple-indicator dilution (MID) technique to study the sinusoidal
transfer constants for HA in the single-pass in situ perfused rat liver
preparation at various steady levels of bulk unlabeled substrate. This
method entails the introduction of a bolus injection into the inflowing perfusate of both
[3H]HA and a set of
noneliminated reference indicators against a set of background
steady-state concentrations of unlabeled hippurate. We used
51Cr-labeled RBC as a vascular
reference, 125I-labeled albumin
and [14C]sucrose as
high and low molecular weight interstitial references, respectively
(14), and
2H2O
as a cellular reference (30). By kinetic analysis of the outflow
profile of the study substance,
[3H]HA, in relation to
those of the simultaneously introduced references, we obtained
estimates of parameters describing unidirectional tracer cellular
influx and efflux, using the barrier-limited model of Goresky et al.
(15). Competition of HA uptake by benzoate and
L-lactate was further examined.
 |
MATERIALS AND METHODS |
Materials
Unlabeled HA, benzoyl chloride, and bovine serum albumin (BSA; fraction
V) were purchased from Sigma Chemical (St. Louis, MO).
[2-3H]glycine (sp act
43.4 Ci/mmol) was obtained from DuPont (Markham, ON).
[51Cr]sodium chromate
(1.61 mCi/mg) and
2H2O
(>99.98% pure) were purchased from Merck Frosst (Montreal, PQ).
[3H]sucrose (11.9 Ci/mmol) was obtained from New England Nuclear (Boston, MA). All
reagents used were of glass-distilled high-performance liquid
chromatography (HPLC) grade or the highest purity available (Fisher
Scientific, Mississauga, ON).
Synthesis of [3H]HA.
[3H]HA was synthesized
from benzoyl chloride and
[3H]glycine under
aqueous and alkaline conditions (19).
[3H]glycine (11.5 nmol
or 500 µCi) was dissolved in 100 µl of 0.05 N sodium hydroxide. To
this, 150 µl of an ethereal solution of benzoyl chloride (80 µM)
were added, and 200 µl of 0.1 N sodium hydroxide were subsequently
added drop by drop. After the reaction mixture was stirred for 1 h, 200 µl of 0.1 N hydrochloride and 100 µl of chloroform were added. The
aqueous phase (top layer) was removed for purification by HPLC. After
purification, the radiochemical purity estimated for HA by HPLC was
>98%.
Distribution of HA Between Chloroform and Perfusate
The distribution of HA into Krebs-Henseleit bicarbonate solution (KHB)
and chloroform was studied at HA concentrations of 3, 30, and 300 µM.
Because the equilibrium distribution ratio was expected to be low, any
impurity of [3H]HA,
albeit representing a very small percentage of the total radioactivity,
posed a complication for quantitation. For this reason, only unlabeled
HA was used in the determination of the distribution ratio. The
partitioning of HA in 20 ml of KHB (pH 7.4) and 20 ml of chloroform was
studied. After the mixture was shaken in a capped 50-ml test tube and
subsequently centrifuged, 10 ml of the chloroform phase was removed and
assayed for HA. The lowest concentration of HA in chloroform (3 µM)
was below the detection limit of the HPLC procedure. For the other two
concentrations (30 and 300 µM), the ratio of chloroform to buffer was
found to be constant (0.0001 ± 0.000015;
n = 3).
Protein Binding of HA and Distribution into RBC
Plasma protein binding.
The binding of HA to albumin was studied with ultrafiltration (10,000 mol wt cutoff, filter no. YM10; Amicon). Hippurate (0.5-500 µM)
containing [3H]HA was
prepared in perfusate plasma (5% BSA) and subjected to ultrafiltration
at 1,000 g (M2-J centrifuge; Beckman,
Mississauga, ON) for 20 min at room temperature. The total HA
concentration in plasma (Cp) was
determined by HPLC and liquid scintillation spectrometry, and the
unbound concentration (Cp,u) in
the ultrafiltrate was quantified by virtue of the radioactivity and the
specific activity of the original plasma sample. The binding constants were initially estimated by expressing the concentration ratio of bound
to free, or (Cp
Cp,u)/Cp,u,
vs. the free HA concentration, Cp,u. Fitting of the data was
subsequently performed by regression of the following expression, for
one class of binding sites
|
(1)
|
where
[Pt] is the total protein concentration,
n is the number of binding sites, and
Kd is the binding
dissociation constant, or the reciprocal of
Ka, the
association constant for binding.
Distribution into RBC.
The distribution of HA into RBC was investigated by mixing plasma
perfusate (5% albumin) containing varying concentrations of HA (up to
883 µM), [3H]HA, and
[14C]sucrose (a
reference that does not enter RBC) with an equal volume of blank blood
perfusate containing 40% RBC (vol/vol) and 5% albumin. The admixture
resulted in a composition identical to that used for perfusion studies
(20% RBC, vol/vol, and 5% albumin). The samples were incubated at
37°C in a rotating water bath for 30 min. Aliquots of perfusate
plasma solution, before and after admixture, were assayed for
[14C]sucrose and
[3H]HA by liquid
scintillation spectrometry, whereas unlabeled HA was assayed by HPLC.
The concentration ratio of
[3H]HA in RBC to the
unbound [3H]HA in
plasma water,
, was estimated by a formula developed earlier (29),
and was found to be essentially zero.
Binding of HA to intracellular components (tissue binding).
Tissue binding was studied in both liver homogenate (1:5 dilution) and
the 9,000 g supernatant. The liver was
first homogenized with 4 vol of ice-cold KHB (homogenizer by
Ultraturrax T25; Janke & Kunkel IKA-Labortechnik), and then a 1:10
dilution of the homogenate was centrifuged at 9,000 g for 20 min at 4°C to provide the
S9 fraction. Bulk HA and
[3H]HA were added to
the homogenate and S9 supernatant such
that the concentrations of HA varied from 1.1 to 511 µM
(10,000-20,000 dpm/ml
[3H]HA); 1.0 ml of the
homogenate and S9 solution was used
for ultrafiltration (10,000 mol wt cutoff; Centricon; Amicon) at 1,000 g for 20 min at room temperature.
Preliminary investigation showed that the leakage of liver protein in
the ultrafiltrate, prepared in the manner outlined above, was <1% of
the total protein present. Protein was evaluated by the method of Lowry
et al. (24). The radioactivities in homogenate and
S9 before ultrafiltration
(Ct) and in the ultrafiltrate (Ct,u) were measured, and the
concentrations of bulk HA in both the homogenate and
S9 fractions were assayed by HPLC.
Rat Liver Perfusion
Male Sprague-Dawley rats weighing 274-375 g (Charles River, St.
Constant, PQ; livers were 8.3-13.3 g) were used for liver perfusion. The animals were housed in accordance with approved protocols of the University of Toronto Animal Committee,
kept under artificial light on a 12:12-h light-dark cycle, and allowed access to water and food ad libitum. The perfusate contained 20% freshly obtained, washed bovine RBC (Ryding Meats, Toronto, ON), 5%
BSA, and 17 mM glucose (Travenol Labs, Deerpark, IL) in KHB buffered to
pH 7.4. The perfusate was oxygenated with 95%
O2-5% CO2 (Matheson, Mississauga, ON)
and O2 (BOC Gases, Whitby, ON) and
was maintained at pH 7.4 by an online flow-through pH electrode (Orion,
Boston, MA). Perfusion was carried out at 37°C in a single-pass fashion as previously described (5), with perfusate (12 ml/min) entering via the portal vein and exiting via the hepatic vein. The
hepatic artery was ligated.
Single-pass perfusion.
Previous liver perfusion studies had confirmed the lack of removal of
HA when it is formed from benzoic acid; only trace levels of hippurate
were found in bile (5). Moreover, preliminary studies showed that a
constancy in the perfusate outflow and biliary excretion was reached by
20 min after perfusion commenced. Single-pass studies were conducted at
12 ml/min for 60 min for all studies. Only one concentration of HA
(1-930 µM) was used per rat liver. For the first set of
competition experiments, HA (~200 µM) and 20 mM
L-lactate were kept constant in
the inflowing perfusate. For the second set of competition studies, 5 µM HA and benzoate (varying from 10 to 873 µM) were present in the
inflowing perfusate; in this set of studies, the HA in the outflow was
expected to exceed that entering the liver due to its formation from
benzoate.
The inflow and outflow samples were collected at steady state (between
15 and 55 min), and the average (3-5 samples) was used to
determine the input (Cin) and
output (Cout) plasma
concentration of unlabeled HA. Bile was collected from 20 min onward,
at 5-min intervals. At the end of each perfusion experiment, the livers were perfused with 25 ml of ice-cold KHB, removed, weighed quickly, and
homogenized with an equivalent volume of KHB (1:1, wt/vol). The
homogenates were stored at 20°C until analysis.
Multiple-indicator dilution.
A MID dose was introduced into the portal vein 20 min after initiation
of all perfusion studies. Sham experiments (without liver) were
conducted to characterize the dispersion due to the inflow and outflow
catheters. MID was conducted as described previously (13). The
injection mixture (0.23 ml), containing
51Cr-labeled washed bovine RBC
(0.4 ± 0.14 µCi),
125I-labeled albumin (3.7 ± 1.7 µCi),
[14C]sucrose (2.1 ± 2.5 µCi),
[3H]HA (1.9 ± 1.1 µCi),
2H2O
(0.099 ± 0.032 ml), and unlabeled HA, in a composition otherwise identical to that of the perfusate, was introduced into the inflow system by an electronically controlled HPLC injection valve.
Simultaneously, outflow samples were rapidly collected at successive
1-, 2-, and 3-s intervals for a total of 180 s by a fraction collector.
Bile was collected at 5-min intervals after MID injection for the next 40 min. The hematocrit of the blood perfusate and dose was determined for each experiment with the use of a hematocrit centrifuge (MB microhematocrit centrifuge; International Equipment Company Division, Fisher Scientific).
Quantitation of Radiolabels or Stable Isotopes
The 51Cr and
125I radiolabels in blood outflow
perfusate samples (25-200 µl) and in the 1:10 diluted dose were
assayed by gamma counting (Cobra II; Canberra-Packard, Mississauga,
ON); the [14C]sucrose
and [3H]HA in plasma
perfusate (50-200 µl) and in the 1:10 diluted plasma dose were
assayed by liquid scintillation counting (Scintillation Counter 5801;
Beckman), as previously described (13).
2H2O
was assayed by Fourier transform infrared spectrometry (model 1600;
Perkin Elmer, Rexdale, ON) over a frequency interval of 2,300-2,700 cm
1 (30).
Recovery of 51Cr,
125I,
14C, and
3H radiolabels and
2H2O
in outflow samples was virtually complete.
Assay of Unlabeled HA in Plasma and Bile
The concentrations of unlabeled HA in plasma samples and bile were
assayed by HPLC, as previously described (5). The HPLC method was used
for the quantitation of the HA in the
S9 fraction and liver homogenate.
Data Treatment
For the MID data, outflow radioactivity for each indicator was
expressed as a fraction of the radioactivity of injected mixture per
milliliter of blood (13). The concentration of radiolabels at the end
of the collection (180 s) was <0.1% of peak values. Recoveries were
calculated as the product of the time integrals of the fractional
recovery and blood flow. Fractional recovery integrals were
approximated by summing the products of fractional recoveries and
sample intervals; fractional recovery activity-time integrals
[area under the curve (AUC)] and integrals of the product of fractional recovery and time [AUC at midintervals
(AUMC)] were calculated similarly (13, 32). The ratio of AUMC to
AUC yielded the mean transit time.
Modeling.
A scheme (Fig. 1) was developed to describe
the kinetic events underlying the disposition of HA in the perfused rat
liver preparation. HA in the plasma space is present as bound and
unbound forms, and only the unbound HA in the plasma compartment
(assumed to be the same for sinusoidal plasma and interstitial space)
is assumed to exchange with that in the hepatocellular compartment. Rapid equilibrium between bound and unbound forms was assumed. It
should be noted that, since albumin is excluded from part of the Disse
space (14), the space of distribution for bound HA is identically
diminished. Transfer rates depend on the rate constants for entry into
(k1) and efflux
from
(k
1) the
hepatocytes, as defined in Table 1. The
rate constants, when multiplied by the accessible cellular water space
(Vcell), yield the
permeability-surface area products for transport
(PinS or
PoutS ). The rate
constant for HA removal solely by excretion
(kseq) is
virtually zero and is neglected in the modeling of the
[3H]HA curve. With
these assumptions, preliminary studies showed that an adequate fit to
the data was attained with the barrier-limited model of Goresky et al.
(13, 15, 32).

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Fig. 1.
Schematic representation of hippuric acid (HA) uptake at the level of a
single sinusoid. Equilibrium is assumed to exist between bound and
unbound forms of HA in plasma and tissue. Rate constants for influx
(k1) and efflux
(k 1)
have been defined in reference to the accessible cell water volume
(Vcell; see Table 1).
Sequestration and thus the constant denoting removal
(kseq) are zero
for HA. Cp,u and
Cp,b are unbound and bound
concentrations of tracer HA in the sinusoid, respectively, and
Ct,u is the unbound concentration
of tracer HA in the cell. Hatched area represents endothelial cells.
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Table 1.
Interrelationships between influx, efflux, and sequestration
coefficients and their physical equivalents
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Superposition of noneliminated references and appraisal of influx
and efflux coefficients.
Superposition of the noneliminated reference indicators (labeled
albumin, sucrose, and
2H2O)
was performed. We used the relationship between the outflow recovery
(concentration/dose) for the noneliminated tracer,
C(t), or the
convolution of the organ transport function
h(t) with the outflow profile
[Ccath(t)] of the sham experiment that defines the dispersion of the inflow and outflow catheters (for details, see
APPENDIX, Eqs.
A1-A3). The
procedure provided values of
t0, a common
large vessel transit time, and
, a space ratio. For the interstitial
space tracers 125I-albumin and
[14C]sucrose,
is
the ratio of the accessible albumin or sucrose Disse space to the
sinusoidal plasma space, and for
2H2O
this ratio is the sum of the accessible Disse and hepatic cellular
water spaces and that in the sinusoid (in RBC and plasma). Equation A3 indicates that, after
t0 (transit time
of large vessel) and the transit time of the input and collecting
systems, each point on the
125I-albumin,
[14C]sucrose, or
2H2O
curves will be delayed in time, relative to the corresponding point on
the RBC curve, by the factor (1 +
), and its magnitude will be
correspondingly attenuated by the factor 1/(1+
).
[3H]HA outflow dilution
curves.
We used the relationship between the outflow recovery
(concentration/dose) for the diffusible substance (HA),
Cdiff(t),
or the convolution of the organ transport function,
hdiff(t),
with the outflow profile of the inflow and outflow catheters,
Ccath(t) (see APPENDIX, Eq. A10), and that of the noneliminated reference, sucrose
[Csuc(t),
see Eq. A1]. A quantitative
analysis of the [3H]HA
outflow profile was carried out with a model developed previously (15,
32). Because binding of HA to RBC is negligible, the hypothetical
reference that described the extracellular behavior of
[3H]HA was constructed
based on the unbound fraction of HA in plasma (fu) and very rapid exchange
between bound and free forms (Eq. A5). A similar strategy was used for salicylamide
sulfate (39) and the glutathione conjugate of bromosulfophthalein (13).
The calculated parameter
ref
(Eq. A5) provides a value for the
interstitial space ratio of a hypothetical reference that, outside the
cells, behaves in a manner identical to that for HA. It is expected to change with HA concentration, as binding of HA to albumin
(fu) changes. The unbound
fraction fu is calculated with the
known binding parameters
Ka and
n obtained from the binding studies (40)
|
(2)
|
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The theoretical reference transport function may then be
appropriately related to that for sucrose (Eq. A7) in describing the extracellular behavior of HA.
The coefficient for intracellular sequestration,
k'seq,
was set to zero. The organ transport function for HA was then
calculated with Eq. A8. The rate
coefficients for influx,
fuk
',
and efflux,
k'
1,
defined in Table 1 were provided by the fitting procedure. The first
term represents material that propagates through the system without
entering the liver cells, or the throughput component. The second term
represents material that enters the liver cells and returns later and
exits via the vascular pathway, or the returning component. As
defined by Goresky et al. (15),
k1 and
k
1 are the
permeability-surface area products for influx and efflux across the
hepatocyte membrane, respectively, per milliliter of cell water
(Vcell) (see Table 1).
To obtain
k1, the product
fuk1
'
is divided by fu
(Eq. 2) and the space distribution
ratio,
', or the ratio of
Vcell to the extracellular
distribution space for HA (Eq. A9). Alternatively, the influx
permeability product
PinS was obtained
with Eq. A11. Normally, the influx
parameters are related to the logarithmic average of the unbound input
and output concentrations (12, 13, 40). Because there was a lack of
hippurate elimination, the unbound concentration in the plasma
(Cu) is constant throughout the
sinusoids and is given by
finCin,
the product of the unbound fraction in input plasma
(fin) and the steady-state input
concentration (Cin), or
foutCout,
the product of the unbound fraction in output plasma
(fout) and the steady-state
output concentration (Cout).
The efflux rate constant,
k
1, was
obtained by dividing
k'seq
by ft, the unbound fraction of HA
in liver tissue. The unbound tissue concentration,
Ct,u, was calculated from the
tissue partition equilibrium ratio,
k1/k
1,
which equals
Ct,u/Cu.
Superposition and MID fitting procedures.
From the fractional outflow recovery curve of the vascular reference
(the labeled RBC curve), the transport function of the injection and
collection system of the outflow profile for the sham experiments
conducted with injection of an MID dose into the inflow and outflow
catheters, without the presence of a liver, was deconvoluted (13). A
linear flow-limited transformation of the deconvoluted RBC curve was
then carried out to generate a calculated first pattern for each
diffusible reference, by selection of trial values for the ratio of the
extravascular to vascular distribution spaces and of
t0, the common
large-vessel transit time. The resulting curve was convoluted with the
system transport function. The generated diffusible reference curve
(for labeled albumin, sucrose, or water) was compared with that
obtained experimentally, and the parameter values were repetitively
refined until a best fit was obtained using a least-squares procedure
(International Mathematics Statistical Library, Visual Numerics,
Houston, TX). The classical weighted least-squares
approach to parameter estimates, as discussed by Landaw and DiStefano
(22), was used as the criterion for fitting. A weighting strategy was
carried out according to counting statistics noise, assuring an error
variance proportional to the magnitude of the observation (7, 22). The
Jacobian matrix (matrix of sensitivities) obtained from the fitting
program was used to calculate variances and covariances of the fitted parameters. The square roots of the variances and the standard deviations of the fitted parameters for each experiment represented the
uncertainty in the parameter estimate.
With these values in hand, a similar process was used to gain best fit
values for influx and efflux coefficients for HA. The outflow dilution
data were fitted to Eq. A8 by
variation of
fuk1
', and
k'
1,
as described previously (13, 32), using the same fitting procedure as
above. The tracer HA outflow profile was further resolved into
throughput and exchanging (returning) components. The dependence of the
parameters on HA concentration was taken into account in that the
nonlinear binding to albumin over the concentration range was
considered (the fraction of unbound HA increased from 0.39 to 0.56 when
the input concentration of HA varied from 1 to 930 µM).
Statistics
All data are means ± SD. Student's
t-test statistic was used, and a
P value
0.05 was viewed as
significant.
 |
RESULTS |
Protein binding of HA.
The binding of HA to albumin was concentration dependent. One class of
binding site was found (n = 1.03),
with a Ka of 2.1 × 103
M
1 (Fig.
2). Within the concentration range used for
the MID studies, the unbound fraction of HA in plasma varied from 0.39 to 0.56. The extent to which HA was bound to liver homogenate or
S9, however, did not vary with HA
concentration (1.1 to 500 µM); values for the unbound fraction of HA
in diluted homogenate and S9 were 1 ± 0.012 and 0.99 ± 0.02 (n = 5), respectively. The values suggest a lack of binding of HA to tissue
proteins.

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Fig. 2.
Binding of HA to 5% bovine serum albumin. Plot of concentration ratio
of bound-to-free HA vs. concentration of free HA. Data suggest the
presence of one class of binding sites. Line is predicted based on
fitted constants.
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Hepatic extraction and biliary excretion of HA.
When perfusate containing increasing bulk plasma concentrations of HA
(1.3 to 930 µM) was used for perfusion, the steady-state hepatic
extraction ratio of unlabeled HA remained virtually zero. Only trace
amounts of HA were found in bile; the biliary excretion of HA was
0.35 ± 0.14% of the total dose. Given the extremely low
excretion rate of HA, the use of a model without sequestration appeared
justified.
Linear superposition by use of the delayed-wave model.
Recoveries of labeled RBCs, albumin, sucrose, and
2H2O,
including [3H]HA, in
hepatic venous blood were complete within experimental errors.
Representative outflow profiles for the labeled substances injected
into the portal vein of the liver are shown in Figs. 3 and 4. The
labeled RBC emerged first and reached the highest and earliest peak;
the RBC outflow curve had the steepest upslope, and the downslope
decayed most rapidly. The
125I-albumin curve rose slightly
less quickly and decayed with a slightly reduced slope, showing a lower
and later peak. In comparison to the labeled albumin curve, the
[14C]sucrose curve
showed a slightly more delayed upslope, a slightly lower and later
peak, and a more prolonged downslope. The greatest dispersion was seen
with
2H2O,
whose upslope and downslope were very delayed and whose peak occurred
much later with a much lower magnitude, due to its permeation of the
cellular as well as vascular and interstitial spaces.

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Fig. 3.
Linear plots of fractional recovery of
[3H]HA and
noneliminated references in multiple-indicator dilution studies
conducted at various steady-state input plasma concentrations of
hippurate. RBC, red blood cells.
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Fig. 4.
Semilogarithmic presentation of tracer
[3H]HA and
noneliminated reference outflow profiles at various steady-state plasma
concentrations of hippurate; data are same as those in Fig. 3. Outflow
profiles were resolved into throughput (dotted area) and returning
components. Reference curve to which the HA curve is related is
outlined; the corresponding space of distribution is slightly smaller
than that for labeled sucrose due to binding to albumin.
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Superposition of the noneliminated reference indicator curves onto the
labeled RBC curve with the deconvolution procedure provided the
optimized parameters
t0 and
(Table
2). The average t0 value (3.7 ± 1.3 s) was similar to those obtained for other perfused livers
(12, 13). The
values for the interstitial substances (for labeled
albumin and labeled sucrose) and for
2H2O
(
Alb = 0.65 ± 0.22,
Suc = 1.1 ± 0.5, and
= 5.2 ± 1.5) were similar to those estimated in a similar fashion
in other perfused rat liver preparations (12, 13). After the approximation of the AUMC and AUC for the noneliminated indicators with
cubic splines, the average mean transit times were estimated by moment
analysis and were converted to their respective volumes after
multiplication by appropriate flows (Table 2). The mean transit times
for the noneliminated indicators were generally similar to those
obtained in other perfused rat liver MID studies.
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|
Table 2.
Mean transit times of noneliminated reference indicators and their
distribution volumes obtained with superposition and splining
procedures for hippuric acid in multiple-indicator dilution studies
in perfused rat liver
|
|
Evaluation of MID results: outflow profile for HA.
The rising upslope of
[3H]HA was slightly
delayed with respect to that of labeled albumin, but it slightly
preceded the labeled sucrose curve (Fig. 3). The
[3H]HA curve crossed
over the labeled sucrose curve, then peaked lower and earlier than the
labeled sucrose curve, as expected, due to binding of HA to albumin.
During its decay, the
[3H]HA curve again
crossed over the labeled sucrose curve and exhibited a more delayed
downslope (Fig. 4). Fits to representative experiments are shown in
Fig. 4. The tracer
[3H]HA outflow profile
was further resolved into the throughput and exchanging (returning)
components. The throughput component increased from 40 to 60% of the
total dose over the unbound concentration range studied (Fig.
5).
The optimized parameters obtained by fitting
rel, the influx coefficient
fuk1
',
and the cellular efflux coefficient
k'seq are summarized in Table 3.
PinS estimated with
Eq. A11 was 3.5 ± 0.6 times that
of the plasma flow rate (0.017 ± 0.002 ml · s
1 · g
1)
at the lower HA concentration used, and these values decreased with
increasing concentration, demonstrating saturability (Fig. 6A). The
corresponding k1
values were also concentration dependent (Fig.
6B). Fitting of these values to
Vmax/(Km + Cu) yielded the apparent
constants for uptake: with
PinS,
Km = 162 ± 53 µM and Vmax = 19 ± 6 nmol · s
1 · g
1,
and with k1,
Km = 182 ± 60 µM and
Vmax = 12 ± 4 nmol · s
1 · g
1
(mean ± SD of parameter estimate); a slight but insignificant difference in these estimates existed due to the reliance of
k1 on
Vcell and subsequently
'.
Saturation was also displayed for PoutS and
k
1; the
latter was equal to the efflux coefficient, since the unbound fraction
in tissue, ft, was found to be
unity (Fig. 7; Table 3). Fitting these
values to the estimated tissue unbound concentration,
Ct,u, yielded very similar kinetic
constants (Km = 330 ± 140 and 390 ± 190 µM;
Vmax = 42 ± 16 and 29 ± 13 nmol · s
1 · g
1)
for efflux. The tissue equilibrium partitioning ratios,
k1/k
1, were slightly lower than unity, and the values were similar to that
found in the liver of the hairless guinea pig (24). The values were
highest at the lower HA concentrations (average value of 0.82 ± 0.19) but gradually decreased with increasing concentration (Fig.
8).
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|
Table 3.
Optimized parameter values derived for hippuric acid in rat liver
perfusion studies, from the fitting procedure
|
|

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|
Fig. 6.
Plots of influx permeability-surface area product
(PinS )
(A) and influx rate constant
(k1)
(B) as functions of unbound plasma
concentration of HA. Lines are fitted lines that yield similar
Km and
Vmax for HA
influx. See text for details.
|
|

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|
Fig. 7.
Plots of efflux permeability-surface area product
(PoutS )
(A) and
k 1
(B) as functions of calculated
tissue unbound concentration of HA. Lines are fitted lines that yield
similar Km and
Vmax for HA
efflux. See text for details.
|
|

View larger version (11K):
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|
Fig. 8.
Plot of the theoretical tissue partition coefficient,
k1/k 1,
as a function of unbound plasma concentration of HA.
|
|
Interactions with L-lactate and
benzoate.
The hepatic transfer of HA in the presence of
L-lactate (20 mM) and benzoate
(from 10 to 873 µM) is summarized in Table
4. The values of
PinS for HA uptake
at ~180 µM (for controls see Table 3, preparations
11-13) were statistically different from those
in the presence of L-lactate,
although the changes in
PoutS, k1 and
k
1 were
not significant. In the presence of benzoate (10 µM), the outflow HA
concentrations increased to 13 µM, whereas for benzoate
concentrations >200 µM, HA outflow concentration varied from 81 to
103 µM, due to HA formation from the various concentrations of
benzoate. The accrued HA concentration was expected not to evoke
changes in transport, since the influx and efflux parameters had
remained rather constant at input HA concentrations <200 µM (see
Figs. 6 and 7). The changes observed were therefore induced by
benzoate. PinS and
PoutS and
k1 and
k
1 for HA uptake were decreased in the presence of benzoate (see Table 3, preparations 1-10 for controls).
View this table:
[in this window]
[in a new window]
|
Table 4.
Interaction between hippuric acid with L-lactate and
benzoate in single-pass rat liver perfusion MID studies
|
|
 |
DISCUSSION |
Hippuric acid, similar to its precursor benzoic acid (5), is found to
exhibit poor binding properties to albumin
(n = 1, Ka = 2.1 × 103
M
1) and does not
distribute into RBC. As expected with these binding constants, the
unbound fraction of HA increased only slightly, from 0.39 to 0.56, over
the wide input plasma concentration range of HA studied (1-930
µM). Binding of HA to intracellular (tissue) components was also
found to be absent.
A negligible extraction ratio of HA was observed, confirming the
previous observation that HA was poorly excreted by the perfused rat
liver preparation (5). To gain insight into the transfer processes, we
estimated the transfer coefficients and transfer rate constants from
the MID experiments, since the events underlying the handling of
hippurate were revealed in its outflow behavior in relation to the
outflow behavior of other reference materials. We found that the
transport parameters for influx and efflux (transfer clearances or rate
constants) were relatively constant for HA input concentrations below
200 µM but eventually decreased with increasing concentration. The
transfer processes are, however, quite rapid for hippurate:
PinS was three to four times that
of the plasma flow rate at low concentrations and one to two times that
of the plasma flow rate at higher concentrations. Efflux was equally as
fast (Table 3). The lack of hepatic excretion of HA is due purely to
its poor candidacy for excretion and is not a result of poor
penetration.
Consistently, the magnitude of
PinS (or influx
clearance per gram liver) and
k1 (Fig. 6) and
their efflux counterparts
PoutS and
k
1 (Fig.
7) was reduced with rising concentrations. The increasing throughput
component (Fig. 5) and the declining partition coefficient (Fig. 8)
conform to the assumption that a carrier protein is involved, since
saturation is evident (12, 13). The alternate mechanism of passive
diffusion, however, is untenable, since the distribution ratio
(equilibrium partitioning of drug into chloroform and buffer at pH 7.4)
was extremely low (0.0001). A similarly low value was also obtained by
Lanman et al. (23). The
Km for influx is
quite high (160-180 µM), and this explains why the transport
remained virtually first order for input plasma concentrations of 200 µM (corresponding to 100 µM unbound concentration). The
Km for efflux is
even higher (330-390 µM). The rate of distribution of hippurate
into the tissue thus appears to be limited by the sinusoidal permeation
of HA molecules from blood to tissue.
That carrier proteins are involved in the transsinusoidal transfer of
HA was evident, since the uptake and efflux displayed saturation. The
anion transport protein oatp expressed in HeLa cells (34) was, however,
not involved in hippurate or benzoate uptake (K. S. Pang and A. W. Wolkoff, unpublished observations). There was demonstrable competition
by L-lactate and benzoate
(10-873 µM), which depressed
PinS and
PoutS. The reduction
in PS products for HA influx and efflux by
L-lactate suggests the putative
role of the hepatic monocarboxylate transporter, MCT2 (9, 10); the
natural substrate is likely to be
L-lactate (8). The interaction between L-lactate and benzoate
has been thoroughly studied in the cloned and expressed MCT
transporter, MCT1, in hamster and rabbit intestine (9, 10, 36, 37).
Reduction of L-lactate but not
D-lactate transport by benzoate
and inhibition by
-cyanocinnamide were observed (37).
Carrier-mediated transport of benzoic acid was found to occur within
Caco-2 cells; a pH dependence was further identified (38). Fast
disappearance of benzoate from peritoneal fluid, suggestive of
carrier-mediated uptake by the peritoneum, was recently reported (28).
All of these findings point to transport of benzoic acid by MCT. The
present data suggest that hepatic transport of the carboxylates by this
transporter may be extended to hippurate in the rat liver.
 |
APPENDIX |
The model used for interpreting the data in this study was the
barrier-limited, space-distributed, variable transit time model developed by Goresky et al. (15). It describes the relationship between
the dose-normalized outflow profiles for the substance under study,
Cdiff(t),
and those of the interstitial reference substances (sucrose or
albumin). Because HA binds to plasma proteins but not RBC, a
hypothetical reference accounting for partial binding, similar to that
proposed for enalaprilat (32), is defined with respect to the
proportion bound to labeled albumin and that which is unbound. In the
absence of binding and uptake, the latter would behave like labeled
sucrose.
The single path (or single sinusoid) model.
The sinusoid is thought to be a single pathway with adjacent sheets of
hepatocytes, from which the irreversible biliary excretion occurs.
Within the sinusoid, with the small lateral dimensions, diffusion in
the lateral direction is assumed to be instantaneous; the sinusoid is
so long, however, that diffusion in the longitudinal direction will not
contribute significantly to transfer from entrance to exit over the
time scale involved, and this is therefore neglected. With these
assumptions, space can be described by a single variable, x, denoting position along the
sinusoid flow path, and it is possible to find an analytical solution
in time and space describing the behavior of tracer within the
vasculature and tissue and at the outflow from a sinusoid.
To evaluate the experimentally obtained outflow profiles, the
dispersion of the injected bolus by the injection apparatus and the
inflow and outflow catheters must be considered, as previously described in detail (13). For example, the experimental sucrose curve,
CSuc(t),
is the convolution of the organ sucrose transport function
(catheter-corrected outflow profile or impulse response), hSuc(t),
with the outflow profile obtained from the apparatus in the absence of
a liver,
Ccath(t)
|
(A1)
|
where
* is the convolution operator. Similarly, for RBC
|
(A2)
|
The
transport functions were computed from the experimental data for
Csuc(t),
CRBC(t),
and
Ccath(t)
by deconvolution, using an algorithm obtained from the National
Simulation Resource in Mass Transport and Exchange, University of
Washington, Seattle, Washington (1, 2).
The parameters of linear superposition according to the flow-limited
model of Goresky et al. (15), i.e., the interstitial to vascular
distribution spaces,
, and the common large-vessel transit time
t0, were found by
first calculating the RBC transport function,
hRBC(t),
by deconvolution as mentioned above and then calculating the organ
sucrose transport function,
hSuc(t),
from the organ RBC transport function,
hRBC(t),
according to the following equation
|
(A3)
|
A calculated sucrose outflow profile is then found by
convolution according to Eq. A1, which
is then fitted to the experimental outflow profile,
CSuc(t),
by a nonlinear least-squares procedure.
Uptake, release, and sequestration of hippurate was evaluated by use of
the barrier-limited space-distributed variable transit time model
developed by Goresky et al. (15). This model allows the determination
of the mass transfer coefficients (Table 1) by comparing the outflow
profile of the substance under study (HA) with appropriate reference
indicators that are not taken up by hepatocytes. Because HA binds to
albumin and is partly excluded from the interstitial space, none of the
experimental reference indicators is directly useable for the modeling
process, and a theoretical reference transport function was
constructed, as follows
|
(A4)
|
where
ref is the ratio of
extravascular to vascular distribution space of HA. The value of this
ratio is
|
(A5)
|
where
fu is the unbound fraction of HA
in plasma.
Because sucrose was used as a reference indicator, the appropriate
reference transport function was calculated as follows (32)
|
(A6)
|
where
|
(A7)
|
From
above, the ratio (1 +
ref)/(1 +
Suc) or the ratio of the
total sinusoidal plasma plus interstitial spaces of distribution for
HA, in relation to that for labeled sucrose, further defines (1 +
rel), which was used to
describe the appropriate interstitial space reference for the data. The
organ transport function for HA was then calculated according to the
barrier-limited space-distributed variable transit time model, using
the following equation
(13)
|
(A8)
|
where
k1 and
k'
1
are transfer coefficients for entry into and efflux from the
hepatocytes, and
k'seq
is the sequestration coefficient describing removal of HA, which was
set to zero in the present case;
' = (1 +
ref)
and
= x/vF,
where x is the distance and
vF is the linear
velocity of sinusoidal blood. The ratio of cellular to extracellular
distribution spaces for HA,
', is obtained from
|
(A9)
|
where
is the ratio of Vcell to
plasma water space (Vp) and
ref is the ratio of the
extravascular to the vascular distribution space of HA
(Eq. A5).
VSuc is sinusoidal sucrose space,
and Vcell and
VSuc are estimated from their mean
transit times.
The calculated HA outflow profile,
CHA(t),
is obtained by convolution of
hHA(t)
with the outflow profile obtained from the apparatus in the absence of
a liver,
Ccath(t)
|
(A10)
|
This
was fitted to the experimental data for
CHA(t)
by varying the parameters
fuk1
',
k'
1,
and
rel. From the fitted value
of
fuk1
',
k1 was calculated
using
', obtained using Eq. A8 and Eq. A10.
Finally, PinS was
obtained from the fitted value of
fuk1
',
as follows
|
(A11)
|
 |
ACKNOWLEDGEMENTS |
We thank Dr. Allan W. Wolkoff of the Marion Bessin Liver Research
Center, Albert Einstein College of Medicine, Bronx, NY, for kind
assistance in the uptake studies of hippurate by oatp in HeLa cells.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grant
GM-38250, the Fast Foundation, and the Medical Research Council of
Canada (MT-11228).
This work was presented in part at the Annual Meeting of the American
Society of Pharmacology and Experimental Therapeutics, San Diego, CA,
1997.
Present address of T. Yoshimura: Dept. of Drug Metabolism and
Pharmacokinetics, Eisai Tsukuba Preclinical Research Laboratories, Tsukuba City, Ibaraki 300-26, Japan.
Address for reprint requests: K. S. Pang, Faculty of Pharmacy, Univ. of
Toronto, 19 Russell Street, Toronto, Ontario, Canada M5S 2S2.
Received 2 June 1997; accepted in final form 5 August
1997.
 |
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