Fatty acid binding protein is a major determinant of hepatic pharmacokinetics of palmitate and its metabolites

Daniel Y. Hung1, Frank J. Burczynski2, Ping Chang1, Andrew Lewis3, Paul P. Masci1, Gerhard A. Siebert1, Yuri G. Anissimov1, and Michael S. Roberts1

1 Department of Medicine, Princess Alexandra Hospital, University of Queensland, Woolloongabba, Queensland Qld 4102, Australia; 2 Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2; and 3 Institute of Pharmaceutical Sciences, University of Nottingham NG7 2RD, United Kingdom


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Disposition kinetics of [3H]palmitate and its low-molecular-weight metabolites in perfused rat livers were studied using the multiple-indicator dilution technique, a selective assay for [3H]palmitate and its low-molecular-weight metabolites, and several physiologically based pharmacokinetic models. The level of liver fatty acid binding protein (L-FABP), other intrahepatic binding proteins (microsomal protein, albumin, and glutathione S-transferase) and the outflow profiles of [3H]palmitate and metabolites were measured in four experimental groups of rats: 1) males; 2) clofibrate-treated males; 3) females; and 4) pregnant females. A slow-diffusion/bound model was found to better describe the hepatic disposition of unchanged [3H]palmitate than other pharmacokinetic models. The L-FABP levels followed the order: pregnant female > clofibrate-treated male > female > male. Levels of other intrahepatic proteins did not differ significantly. The hepatic extraction ratio and mean transit time for unchanged palmitate, as well as the production of low-molecular-weight metabolites of palmitate and their retention in the liver, increased with increasing L-FABP levels. Palmitate metabolic clearance, permeability-surface area product, retention of palmitate by the liver, and cytoplasmic diffusion constant for unchanged [3H]palmitate also increased with increasing L-FABP levels. It is concluded that the variability in hepatic pharmacokinetics of unchanged [3H]palmitate and its low-molecular-weight metabolites in perfused rat livers is related to levels of L-FABP and not those of other intrahepatic proteins.

clofibrate; pregnancy; multiple indicator dilution; hepatic extraction ratio; cytoplasmic diffusion constant


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INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES

NONESTERIFIED, LONG-CHAIN fatty acids (hereafter referred to as fatty acids) play a vital role in many cellular processes. They are an important source for cellular energy and form the building blocks from which various cellular components are synthesized. Some fatty acids are precursors for biological mediators, whereas others may interact with the cell membrane to regulate various cellular functions. Uptake of fatty acids has been shown to parallel the level of liver fatty acid binding protein (L-FABP) (22).

L-FABP levels are known to be gender dependent, with females showing higher levels than males (25). L-FABP is also altered in various conditions, such as pregnancy and clofibrate treatment (6, 26, 31). Although the variation in the content of various intrahepatic proteins in liver disease has been used to account for the uptake, binding, and metabolism of solutes (20), the influence of intrahepatic proteins other than L-FABP [e.g., microsomal protein (MP), cytochrome P-450 (CYP), albumin (Alb), and glutathione S-transferase (GST)] on the hepatic disposition of [3H]palmitate has been studied to a limited extent. Most studies investigating the fatty acid uptake into the liver have used isolated hepatocytes with some studies using intact livers (26, 38).

In this work, we examined the relationship between the hepatic pharmacokinetics of unchanged palmitate and its low-molecular-weight metabolites in perfused rat liver using the multiple indicator dilution (MID) technique and a selective assay for unchanged [3H]palmitate and its low-molecular-weight metabolites. To explore the relationship between palmitate pharmacokinetics and L-FABP and other intrahepatic proteins, a number of liver models in which protein expression varied was used, namely: male, female, pregnant female, and clofibrate-treated male. As in our previous studies (20, 21), a mixture of two inverse Gaussian density functions and a barrier-limited and space-distributed liver model were used to fit vascular reference data and provide estimates of the extracellular and cellular spaces in the liver. The unchanged palmitate data were then evaluated by fitting the outflow time profiles for unchanged palmitate and the markers with various physiologically based pharmacokinetic models. An integrated slow cytoplasmic diffusion, instantaneous binding, barrier-limited, and two-phase-stochastic distribution model (43) was found to optimally describe the data and to provide estimates of the pharmacokinetic parameters for hepatocellular influx (kin), efflux (kout), diffusion (kd), and elimination (ke) for [3H]palmitate.


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MATERIALS AND METHODS
RESULTS
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Materials

All chemicals including fatty acid-free Alb and clofibrate were purchased from Sigma (St. Louis, MO). [3H]palmitate, [3H]water, and [14C]sucrose were purchased from New England Nuclear (Boston, MA).

Purification of [3H]Palmitic Acid.

[3H]palmitic acid was purified by the Borgstrom ethanol extraction procedure as previously described (13). Neutral organic impurities were removed by extracting the aqueous [3H]palmitic acid several times with heptane. After acidification of the ethanol solution with two drops of 6 N HCl, the radiolabeled lipophilic compounds were extracted with heptane. The organic phase containing the purified [3H]palmitic acid was harvested, evaporated under nitrogen, redissolved in ethanol, and stored at -20°C until used.

Clofibrate-Treated Rat Model

The clofibrate rat model was established following a procedure described elsewhere (5). Briefly, male Wistar rats were fed clofibrate 0.35% wt/wt intermixed with standard chow for 6 days.

Analysis of L-FABP Tissue Levels

L-FABP was purified according to the method of Takahashi et al. (40). Rat L-FABP antiserum was raised in rabbits by intradermal injection of 250 µg denatured L-FABP, followed by two subcutaneous injections of denatured L-FABP (200 µg each) at biweekly intervals. Rabbit plasma was harvested and stored frozen (-20°C) until required.

Rat L-FABP sandwich enzyme immunoassay was carried out using polyclonal antibody to rat L-FABP (1:500 dilution) bound to 96-well activated Titertek ImmunoAssay-Pate microtiter plate (Flow Laboratories, Linbro, The Netherlands). The antibody was bound using 0.05 M sodium carbonate buffer (pH 9.6) overnight at 4°C. The remaining binding sites on the plates were then blocked with 5% BSA (Sigma) in wash buffer (0.05 M phosphate buffer, pH 7.4, with added 0.05% Tween 80 and 0.5 M NaCl) for 1 h at room temperature followed by 3 × 10 min wash steps with wash buffer. Rat liver microsomal extracts were prepared and diluted 1:5 and 1:10 in wash buffer. Diluted extract (50 µl) was dispensed in quadruplicates into microtiter plates and incubated at 37°C for 1 h. The washing procedure was repeated and followed by the addition of 50 µl of rat L-FABP-IgG-horseradish peroxidase (diluted 1:500 with wash buffer) to each well and incubated at 37°C in an oven for 1 h as described previously (1). Horseradish peroxidase substrate, 40 mM of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (Sigma) in 0.1 M citric acid buffer, pH 4.0, with 0.5 M H2O2, was used to prepare a working substrate solution as described by Saunders (35). Substrate working solution (50 µl) was added to each well, and the color reaction was monitored using Dynatech MR 580 plate reader (Dynatech Instruments, Torrence, CA) until A405 nm of 1 µg rat L-FAPP standard gave an optical density of 1.0. The reaction was then stopped with the addition of 50 µl stopping solution of 0.1 M hydrofluoric acid, 0.01 M NaOH, and 0.001 M EDTA (35). Cytosolic proteins were purified by centrifugation and ultrafiltration using Millipore Microcon YM-3 filter devices (3,000 molecular wt CO; Millipore, Bedford, MA).

Determination of MP and CYP Tissue Levels

Approximately 1 g of liver was homogenized for 3-5 min in 2.5 ml of ice-cold 0.25 M sucrose containing 50 mM Tris · HCl buffer (pH 7.4). Homogenates were centrifuged at 3,000 g for 20 min, and the pellets were resuspended in 2.5 ml Tris buffer. The supernatant was centrifuged again at 30,000 g for 1 h. The resulting pellets were resuspended in 2.5 ml of Tris buffer. The MP concentration was determined by the method of Lowry et al. (23). CYP content in MP was estimated from the dithionite-reduced difference spectrum of CO-bubbled samples using the molar extinction difference of 104 mM/cm in absorption at peak position (~450 nm) (28).

Determination of Albumin Tissue Levels

Livers were harvested from rats after MID studies and perfused with a mixed solution consisting of solution A, calcium- and magnesium-free HBSS (Sigma, St. Louis, MO); solution B, 5 mM EDTA (Sigma); and solution C, 10 mM HEPES (Sigma) at 10 ml/min for 10 min to remove the protein and blood from sinusoidal beds. Livers were then homogenized using a tissue blender and centrifuged at 3,000 g for 10 min. For tissue Alb level analysis, a previously described dye-binding method was modified (19). Stock solutions were prepared using the following substances: 50 mM succinate buffer, pH 4.2 (standard buffer); rat Alb (Sigma) solution (0-2.5 mg/ml) in the standard buffer; 0.3 mM bromocresol green (Sigma) dissolved in standard buffer. For Alb standard curves, 780 µl of the sample (750 µl solution A plus 30 µl solution B) and 150 µl of solution C were spectrophotometrically assayed at 570 nm (model RF-5301PC spectrofluorophotometer; Shimadzu). For tissue Alb level analysis, 30 µl of the supernatant obtained from homogenized tissue was used instead of solution B.

Analysis of GST Tissue Level

GST tissue levels were assayed according to Alin et al. (2). The method was based on the reaction between 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione in a sodium phosphate buffer reaction system (pH 6.5, 30°C). Activity was measured spectrometrically at 340 nm (epsilon  = 9,600 M/cm). Briefly, 1 ml total reaction mixture contained 1 mM CDNB dissolved in 100% ethanol, 1 mM glutathione, and 50 µl liver tissue homogenate in assay buffer. The reaction rate for all enzyme activity assays (µmol · min-1 · mg protein-1) was determined by subtracting the background activity rate.

In Situ Rat Liver Perfusions

Studies have been carried out in accordance with the University of Queensland Animal Care Committee. The in situ perfused rat liver preparation used in this study has been described previously (20, 21). Briefly, male, female, and pregnant Wistar rats (300-350 g body wt) were anesthetized by an intraperitoneal injection of 10 mg/kg xylazine (Bayer, Australia) and 80 mg/kg ketamine-hydrochloride (Parnell Laboratories). After laparotomy, animals were heparinized (200 units heparin sodium; David Bull Laboratories) via the inferior vena cava. The bile duct was cannulated with a PE-10 catheter (Clay Adams). The portal vein was cannulated using a 16-gauge intravenous catheter, and the liver was perfused via this cannula with 1% fatty acid-free Alb dissolved in PBS composed of (in mM): 137 NaCl, 2.68 KCl, 1.65 KH2PO4, and 8.92 Na2HPO4, pH adjusted to 7.4 using 0.1 N NaOH. To eliminate the possibility of long-chain fatty acid soap formation, no divalent inorganic cations were present in the perfusate. Before entry into the liver, the perfusate was oxygenated using a Silastic tubing lung ventilated with 100% oxygen. The perfusion system used was nonrecirculating and employed a peristaltic pump (Cole-Parmer). After the initiation of liver perfusion, animals were killed by thoracotomy and the thoracic inferior vena cava was cannulated using a PE-240 catheter (Clay Adams). Oxygen consumption, bile flow, perfusion pressure, and macroscopic appearance were used to assess liver viability.

Bolus Studies

Each liver was perfused at a rate of 25 ml/min. After a 10-min perfusion-stabilization period, aliquots (50 µl) of the buffer solutions containing radiolabel or dye-labeled solutes were administered through the portal vein cannula. In each liver, a maximum of two injections [bolus 1: purified [3H]palmitate (3 × 106 dpm), Evans blue (EB) dye (3 mg/ml), and [14C]sucrose (1.5 × 106 dpm); bolus 2: [3H]water (3 × 106 dpm)] were administered in randomized order. The total perfusion time for each liver was <1 h. Before introduction of the Hamilton syringe injection needle into the flowing perfusate, the injection needle was wiped with a heptane-wetted tissue to remove any adherent [3H]palmitate on the outside of the stainless steel needle. Prior experiments, in which the needle was not wiped with heptane, were found to be associated with slightly higher initial outflow profiles, i.e., the first three outflow data points were significantly higher than the respective [14C]sucrose data points. Thus the heptane cleaning procedure eliminated errors in the outflow sample concentration determination during the initial phase of the outflow concentration-time curves, due to adherent [3H]palmitate. A stabilization period of 10 min was afforded between injections. Outflow samples were collected using a fraction collector over 3.5 min, and aliquots (100 µl) were taken for scintillation counting using a MINAXI beta TRI-CARB 4000 series liquid scintillation counter (Packard Instruments). Aliquots (100 µl) were also removed from the outflow samples for absorption spectrophotometric analysis of EB dye at 620 nm by using a Spectracount plate counter (Packard).

Metabolite Separation

[3H]palmitate was separated from any 3H-labeled fatty acid metabolites by a modification of the Dole procedure (12). Trichloroacetic acid (50 µl; 10% solution) was added to Eppendorf tubes containing 100 µl of effluent sampled from the first bolus and vortexed. Samples were centrifuged for 4 min at 10,000 g using a microcentrifuge (Sigma Laborzentrifugen, Harz, Germany). The supernatant was removed, and the Eppendorf tube was cut at a level just above the pellet. The tube remnant containing the pellet was placed directly into a scintillation vial and 2 ml Ready Safe scintillate was added. The pellet was allowed to dissolve overnight, and BSA-associated radioactivity (representing [3H]palmitate) was determined the following day.

Low-Molecular-Weight Metabolites Outflow Concentration-Time Profile

Low-molecular-weight metabolites of [3H]palmitate were separated from the effluent nonextracted samples by an ultracentrifugation method using Millipore Microcon YM-30 filter devices (30,000 molecular wt CO, Millipore) and counted by a beta liquid scintillation counter (Packard). From the outflow concentration-time profile, the production of area under the curve (AUC) low-molecular-weight metabolites of palmitate (AUCmet) and retention of palmitate metabolites in the liver (MTTmet) were determined. The radioactivity in the filtrate was assumed to represent low-molecular-weight metabolites. A thin-layer chromatography (TLC) assay was employed to determine the relative molecular weight of the radiolabeled signal molecules in the effluent as described previously (7). Briefly, low-molecular-weight radioabeled metabolites were separated using a Merck silica gel-60 plate (Merck, Darmstadt, Germany) and eluted with petroleum ether/ethyl ether/acetic acid (90:8.5:1.5). The retardation factor (Rf) values corresponding to each band in the TLC plate were estimated.

Data Analysis

Detailed description of the theory and modeling methods were reported by Weiss and colleagues (21, 43, 45). Briefly, a mixture of two inverse Gaussian density function with correction for catheter effects was used to estimate the extracellular volume (VE determined by [14C]sucrose or EB dye). A barrier-limited plus space-distributed liver model with correction for catheter effects was used to estimate the cellular water volume (VC, determined by [3H]water). The outflow concentrations for unchanged [3H]palmitate were presented as outflow fraction per milliliter. The resulting outflow concentration-time profiles were analyzed using one of the following physiologically based pharmacokinetic models (illustrated in Fig. 1).


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Fig. 1.   Schematic overview of hepatocellular [3H]palmitate transport and clearance in the pharmacokinetic models FABPc and FABPpm representing the cytosolic and plasma membrane fatty acid binding proteins, respectively; KR and KS, rapidly and slowly equilibrating binding constants, respectively; kin, kout, ke, kd, kon, and koff, representing the permeation, efflux, elimination, diffusion, binding, and unbinding rate constants, respectively.

Well-mixed model. Under the assumption of quasi-instantaneous intracellular distribution equilibrium, i.e., a cellular space that is well-mixed perpendicular to flow direction, the cellular behavior of the solute &fcirc;y(s) can be described as (43)
<A><AC>f</AC><AC>ˆ</AC></A><SUB>y</SUB>(s)=<FR><NU>k<SUB>in</SUB><IT>/&ugr;</IT></NU><DE><IT>k</IT><SUB>in</SUB><IT>/&ugr;+k</IT><SUB>e</SUB><IT>+s</IT></DE></FR> (1)
in which &fcirc;y(s) denotes the inverse Laplace transform of the transit-time density function of a solute in the liver, kout kin/v, and v denotes the cytoplasmic volume of distribution v = VT/VE, and kin, kout, and ke are the influx, efflux, and elimination rate constants, respectively. VT is the apparent tissue distribution space, VE is the extracellular reference space, and s is the Laplace variable. With respect to the assumptions on cellular distribution kinetics, this model is identical with the two-compartment dispersion model (14, 34).

Slow binding model. This model assumes rapid intracellular diffusion, but slow binding of molecules to immobile intracellular sites, with the consequence that the cytoplasmic equilibration process is determined by the binding and unbinding rate constants kon and koff after instantaneous distribution in volume VC. The cellular behavior of the solute &fcirc;y(s) can be described as (21)
<A><AC>f</AC><AC>ˆ</AC></A><SUB>y</SUB>(s)=<FR><NU>(s+k<SUB>off</SUB>)<IT>k</IT><SUB>in</SUB></NU><DE><AR><R><C><IT>s</IT><SUP>2</SUP>(<IT>k</IT><SUB>in</SUB><IT>/k</IT><SUB>out</SUB>)(1<IT>+K</IT><SUB>R</SUB>)<IT>+s</IT>((<IT>k</IT><SUB>in</SUB><IT>/k</IT><SUB>out</SUB>)(<IT>k</IT><SUB>off</SUB><IT>+K</IT><SUB>R</SUB><IT>k</IT><SUB>off</SUB></C></R><R><C><IT>+k</IT><SUB>e</SUB><IT>+k</IT><SUB>on</SUB>)<IT>+k</IT><SUB>in</SUB>)<IT>+</IT>(<IT>k</IT><SUB>in</SUB><IT>/k</IT><SUB>out</SUB>)<IT>k</IT><SUB>e</SUB><IT>k</IT><SUB>off</SUB><IT>+k</IT><SUB>in</SUB><IT>k</IT><SUB>off</SUB></C></R></AR></DE></FR> (2)
in which kin = CLpT /VE, where CLpT is the permeation clearance (CLpT = fuPS, in which PS is the permeability surface area product, fu is the free fraction of solute in the perfusate) and kon and koff represent the intracellular binding and unbinding rate constant, respectively, determining the equilibrium amount ratio KS = kon/koff characterize the slowly accessible pool, KR is the equilibrium amount ratio characterizing the rapidly equilibrating binding sites, and the elimination rate constant ke = CLint/VC is the palmitate metabolic clearance (CLint) normalized per cellular water volume (VC).

Slow-diffusion/bound model. Under the assumption that diffusion in hepatocytes is a rate-limiting function, if only the bound drug is contributing significantly to diffusion, the cellular behavior of the solute &fcirc;y(s) can be described as (43)
<A><AC>f</AC><AC>ˆ</AC></A><SUB>y</SUB>(s)=s+k<SUB>in</SUB><IT>−</IT><FR><NU><IT>k</IT><SUB>out</SUB><IT>k</IT><SUB>in</SUB></NU><DE><FENCE><IT>k</IT><SUB>out</SUB><IT>+</IT><RAD><RCD><IT>k</IT><SUB>d</SUB>(<IT>s+k</IT><SUB>e</SUB>)</RCD></RAD></FENCE></DE></FR> tanh<FENCE><RAD><RCD>(<IT>s+k</IT><SUB>e</SUB>)<IT>/k</IT><SUB>d</SUB></RCD></RAD></FENCE> (3)
in which kd is the effective cytoplasmic diffusion constant, and tanh is the effective thickness of the hepatocytes.

Slow-diffusion/unbound model. Under the assumption that diffusion in hepatocytes is a rate-limiting function and only the unbound drug is diffusing, Eq. 3 for the cellular behavior of the solute &fcirc;y(s) has to be modified as        
<A><AC>f</AC><AC>ˆ</AC></A><SUB>y</SUB>(s)=s+km<SUB>1</SUB>−<FR><NU>km<SUB>2</SUB>km<SUB>1</SUB></NU><DE><FENCE>km<SUB>2</SUB>+<RAD><RCD>km<SUB>d</SUB>(<IT>s+km</IT><SUB>e</SUB>)</RCD></RAD></FENCE></DE></FR> tanh<FENCE><RAD><RCD>(<IT>s+km</IT><SUB>e</SUB>)<IT>/km</IT><SUB>d</SUB></RCD></RAD></FENCE> (4)
in which km1 = kin, km2 = kout/(1 + KR), kme = ke/(1 + KR), kmd = kd/(1 + KR). This modification of parameters reflects the fact that only unbound drug returns to the plasma (km2), is eliminated (kme), and diffuses (kmd) in the cellular space.

Nonparametric estimate of hepatic availability (F) of [3H]palmitate in the various models was determined from the outflow concentration (C) vs. time (t) profile from Eq. 2 using the parabolas-through-the-origin method (extrapolated to infinity) with the assistance of the Moments Calculator version 2.2 program (University of Otago) for Macintosh computer (32).
F<IT>=</IT><FR><NU><IT>Q · AUC</IT></NU><DE><IT>D</IT></DE></FR> (5)
in which F denotes the hepatic availability, Q is the perfusate flow rate, and AUC = int <UP><SUB>0</SUB><SUP>∞</SUP></UP> C(t)dt is the area under the solute concentration vs. time curve and D is the dose of solute administered. All concentrations used were expressed in molar equivalents. Hepatic extraction ratio (E) is equal to 1-F.
MTT=<FR><NU>AUMC</NU><DE>AUC</DE></FR> (6)
AUMC is the area under the first moment curve, and MTT is mean transit time.
CV<SUP>2</SUP>=<FR><NU>&sfgr;<SUP>2</SUP></NU><DE>MTT<SUP>2</SUP></DE></FR> (7)
where CV2 is normalized variance and in which
&sfgr;<SUP>2</SUP>=<FR><NU>∫<SUP>∞</SUP><SUB>0</SUB> t<SUP>2</SUP>C(t)dt</NU><DE>∫<SUP>∞</SUP><SUB>0</SUB> C(t)dt</DE></FR>−MTT<SUP>2</SUP> (8)
where int <UP><SUB>0</SUB><SUP>∞</SUP></UP> C(t)dt is the integral from time 0 to time infinity  of the concentration C(t)-time profile.

Statistical Analysis

All data are presented as means ± SD unless otherwise stated. Statistical analysis was performed using the Tukey's post hoc test or regression analysis where appropriate. Statistical significance was taken at the level of P < 0.05. The model selection criterion (MSC) provided by Scientist (MicroMath Scientific Software, Salt Lake City, UT), a modified Akaike Information Criterion (normalized to the number of data points), is defined by the following formula
MSC = ln<FENCE><FR><NU><IT>&Sgr;</IT><SUP><IT>n</IT></SUP><SUB><IT>i=</IT>1</SUB><IT> w</IT><SUB>i</SUB>(<IT>C</IT><SUB>obs<SUB>i</SUB></SUB><IT>−<A><AC>C</AC><AC>&cjs1171;</AC></A></IT><SUB>obs</SUB>)<SUP>2</SUP></NU><DE><IT>&Sgr;</IT><SUP><IT>n</IT></SUP><SUB><IT>i=</IT>1</SUB><IT> w</IT><SUB>i</SUB>(<IT>C</IT><SUB>obs<SUB>i</SUB></SUB><IT>−C</IT><SUB>cal<SUB>i</SUB></SUB>)<SUP>2</SUP></DE></FR></FENCE><IT>−</IT><FR><NU>2<IT>p</IT></NU><DE><IT>n</IT></DE></FR> (9)
in which i represents individual data point, wi is the weight applied to each concentration data point, n is the number of points, Cobsi is the observed concentration, Ccali is the value predicted by the model, <A><AC>C</AC><AC>&cjs1171;</AC></A>obs is the weighted mean of the observed data, and p is the number of estimated parameters. These parameters were used to compare models regarding the "goodness of fit." The most appropriate model, from a statistical point of view, is that with the largest MSC. Information on parameter statistics was obtained from the approximate coefficients of variation of each estimate and the correlation matrix. A high coefficient of variation and/or correlation between parameters was taken to suggest unreliable estimates.


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MATERIALS AND METHODS
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The mean ± SD (n = 6) liver weight of animals used in the perfusion studies was 9.7 ± 1.3 g for male rats, 12.5 ± 1.2 g for clofibrate-treated male rats, 6.7 ± 0.6 g for female rats, and 12.5 ± 1.7 g for pregnant female rats. Clofibrate-treated male and pregnant female rats showed significantly larger liver weights (P < 0.05) than male and female rats. During the course of perfusion, pregnant female rats had significantly lower bile flow than the other groups (P < 0.05). The mean ± SD bile flow, (n = 6) was 0.47 ± 0.05 µl · min-1 · g-1 liver for male rats, 0.43 ± 0.07 µl · min-1 · g-1 liver for clofibrate-treated male rats, 0.51 ± 0.12 µl · min-1 · g-1 liver for female rats, and 0.27 ± 0.07 µl · min-1 · g-1 liver for pregnant female rats. Hepatic oxygen consumption for all animals was in the range of 1.43 to 1.89 µmol · min-1 · g liver-1 and indicated. Perfusion pressure ranged from 9.33 to 13.12 cmH2O. These parameters were comparable to those reported previously (10, 29).

L-FABP, MP, CYP, Alb, and GST tissue levels for each of the animal models are shown in Table 1. The pregnant female group had a significantly higher L-FABP level than the other groups, whereas the levels of other intrahepatic proteins (MP, CYP, Alb, and GST) remained relatively constant. The L-FABP level among the various models was in the following order: pregnant female > clofibrate-treated male > female > male.

                              
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Table 1.   Comparison of L-FABP, MP, CVP, ALB, and GST tissue level in the various animal models

A TLC assay showed that other radiolabeled solutes in the ultrafiltrate had lower Rf values (0.03-0.11) than palmitate (0.31), consistent with these more hydrophilic solutes being low-molecular-weight metabolites of [3H]palmitate (7).

Table 2 lists results derived from moments for unchanged [3H]palmitate (extracted with trichloroacetic acid) and low-molecular-weight metabolites of palmitate (separated by ultrafiltration) in the various animal models. The pregnant female group showed significantly larger hepatic extraction ratio, AUCmet, MTT, and MTTmet values compared with the other groups, whereas CV2 did not appear to be related to L-FABP levels in any of the animal models. The hepatic extraction ratio, AUCmet, MTT, and MTTmet values followed the same order as the L-FABP levels.

                              
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Table 2.   Comparison of nonparametric moments for [3H]palmitate (extracted with trichloroacetic acid) and low-molecular-weight metabolites (separated by ultrafiltration) in the various animal models

Fig. 2 shows typical normalized concentration-time profiles (normal scale) for unchanged [3H]palmitate, low-molecular-weight metabolites of [3H]palmitate, Alb, and [14C]sucrose. A significant efflux of low-molecular-weight metabolites of [3H]palmitate occurred during the perfusion time.


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Fig. 2.   Typical normalized concentration-time profiles (normal scale) for [3H]palmitate (), low-molecular-weight metabolites produced from [3H]palmitate (open circle ), albumin (black-down-triangle ), and [14C]sucrose (down-triangle). Significant efflux of radiolabeled metabolic products occurred during the time course of this study and [3H]palmitate had a profile similar to albumin, due to high protein binding.

Fig. 3A shows the fits for unchanged [3H]palmitate using either Alb or [14C]sucrose as the extracellular reference for unchanged [3H]palmitate. It is evident that the disposition of unchanged [3H]palmitate is best described by using Alb as its reference, consistent with the high protein binding of palmitate to Alb. It is also evident in Fig. 3B that the outflow-time profiles for unchanged [3H]palmitate in normal male rats, using Alb as the extracellular reference, is better fitted by the slow-diffusion/bound and slow-diffusion/unbound models than either the slow-binding model or well-mixed model (Fig. 1). The slow-diffusion/unbound model is, however, less suitable than the slow-diffusion/bound model, because its extra parameter for fraction unbound in hepatocytes increases the parameter coefficient of variation and correlation between certain parameters. Application of the slow-diffusion/bound physiological pharmacokinetic model (Eq. 3) to describe typical outflow concentration-time profiles for unchanged [3H]palmitate in the various animal models used is shown in Fig. 3C (data weighted, 1/yobs), in which 1/yobs is the data weighting used in nonlinear regression. In general, the peak of the unchanged [3H]palmitate outflow curve is lowest (highest hepatic extraction) for the animal model in which L-FABP level is highest.


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Fig. 3.   A: comparison of the data-fitting results using albumin (solid line) or [14C]sucrose (dotted line) as the extracellular reference for [3H]palmitate (). The lines represent the fits of the profiles to the slow-diffusion model. B: comparison of the data-fitting results for [3H]palmitate () using 4 physiologically based pharmacokinetic models: a well-mixed model (dash-dotted line), a slow-binding model (short dashed line), a slow-diffusion/bound model (solid line), and a slow-diffusion/unbound model (medium dashed line). Albumin was used as the extracellular reference for all models. C: typical data-fitting results for [3H]palmitate in the various animal models , male; open circle , clofibrate-treated male; black-down-triangle , female; down-triangle, pregnant female using the slow-diffusion/bound model.

Table 3 lists the fitting results of kinetic parameters for extracted [3H]palmitate in the various animal models. The CLint, PS, kin/kout, and kd values in all parameters showed a trend to increase with the level of L-FABP. The pregnant female group had significantly larger CLint, PS, kin/kout, and kd values than the other groups. These values were in the same order as L-FABP levels.

                              
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Table 3.   Comparison of kinetic parameters derived from the slow diffusion/bound model fitting for extracted [3H]palmitate with trichloroacetic acid in the various animal models


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study confirms and adds to findings about hepatic palmitate pharmacokinetics reported by Luxon and colleagues (24-26). The present work differs in four aspects. First, we increased the sampling time from 90 to 210 s to increase the tail of the curve so as to better compare the appropriateness of the different possible physiological models (Fig. 1). The analysis suggested that the slow-diffusion/bound model was superior to other models, a result consistent with the modeling strategy used by Luxon and colleagues (24-26). Second, we used a lower concentration of purified Alb in the perfusate (1 vs. 2% Alb) to increase the extraction of palmitate. The use of this lower concentration led to measurable metabolite levels in the perfusate outflow. Third, we used selective extraction procedures, such as ultrafiltration, to accurately estimate metabolite concentrations in the effluent and a trichloroacetic acid fractionation of [3H]palmitate in the effluent samples to measure unchanged [3H]palmitate. Finally, we introduced pregnant animals to further add information about the relationship between L-FABP levels and hepatic [3H]palmitate disposition in the liver in addition to male, female, and clofibrate-treated male animals used by Luxon and colleagues in an earlier study (25).

A TLC assay (7) was used to confirm that the radioactivity in the ultrafiltrate was not [3H]palmitate. High-molecular-weight metabolites, such as very low-density lipoprotein (VLDL), would be retained by the filtration membranes and not be detected in the ultrafiltrate. Results published by Burczynski et al. (7) showed that the radiolabel is not necessarily in the 9-10 position of [3H]palmitate, as suggested by the manufacturer, but apparently it can be scattered throughout the molecule. Thus the radiolabel could be found in many different metabolic hydrophilic products. Therefore, the radiolabeled solutes in the ultrafiltrate were generally called low-molecular-weight metabolites in this work.

The time frame for fatty acids to be converted to triglycerides and phospholipids and finally to be integrated with free cholesterol and cholesteryl esters as VLDL has been reported previously. Charlton and colleagues (9) have shown that apolipoprotein B (apoB)-100 (used as a marker for VLDL) increased linearly up to 300 min and there was ~25% enrichment of [14C]leucine in apoB at 120 min (earliest time point), suggesting that the time period for production of VLDL is long. Another report (11) also showed that VLDL production peaked between 60 and 90 min. Because our MID study was completed within 4 min for [3H]palmitate, the assumption is that a negligible amount of radiolabeled VLDL is produced during this short time frame.

A major finding in this study is that both the production of low-molecular-weight metabolites of palmitate and retention of palmitate metabolites by the liver are related to L-FABP. Earlier studies used conditions (25) or methods (17) that did not allow the relationship between metabolite production and L-FABP to be explored. Fatty acids are eliminated in the liver through conversion to VLDL or oxidation by the microsomal omega -oxidation (CYP4A-mediated hydroxylation), peroxisomal beta -oxidation, and mitochondrial beta -oxidation systems (33, 37). Whereas the L-FABP level in the pregnant female group was found in this work to be significantly higher than in other groups, the levels of the other intrahepatic proteins (MP, CYP, Alb, and GST) were similar for all groups (Table 1). Although we have shown that MP and CYP are major determinants for the hepatic CLint of cationic drugs (20, 21), the present work confirms that L-FABP, rather than other intrahepatic binding proteins, is the major determinant of hepatic palmitate and its low-molecular-weight metabolites pharmacokinetics. This is partly due to the fact that the microsomal omega -oxidation is only a minor metabolic pathway (33). The increase in CLint, AUCmet, and MTTmet for unchanged [3H]palmitate with increasing L-FABP levels (Fig. 5, B-D) therefore highlights the role of L-FABP in palmitate disposition. These findings and the higher metabolic rate of unchanged [3H]palmitate in the clofibrate-treated male group are consistent with conclusions reached by Luxon et al. (26) using different conditions. We also recognize that as CLint is a product of ke and VC; higher CLint values for the clofibrate-treated male and pregnant female groups may reflect the larger livers as is evident by higher VC values.

One of the novel aspects of this work is the finding that a slow-diffusion model better describes the hepatic distribution of [3H]palmitate than a slow equilibrating model with immobile intracellular binding sites. In general, the cytoplasmic distribution of a solute after crossing the cell membrane can be described with at least three modeling approaches (Fig. 1): 1) the well-mixed model (barrier-limited tissue distribution model) is conventionally used in most organ models of solute disposition, such as the two-compartment dispersion model (14, 15), assuming a quasi-instantaneous tissue equilibration due to rapid diffusion and binding; 2) the slow-diffusion/bound model (27), which assumes a slowing of diffusion of the solute in the cell due to instantaneous binding to mobile carrier proteins; and 3) the slow-binding model (43, 44), which assumes slow binding to cellular sites after instantaneous distribution throughout the cytosol. In the present work, we also used a slow-diffusion/unbound model to fit the outflow curves, which is similar to the slow-diffusion/bound model except that a rapidly equilibrating binding and dissociation between L-FABP and palmitate is considered. An appropriate definition of the appropriate pharmacokinetic model is aided by the choice of the extracellular reference solute used in the modeling process. Goresky et al. (16) suggested that the extracellular reference should act in every way identically to the solute of interest (i.e., an identical volume of distribution in the space of Disse) except for liver elimination. Our present results suggest that Alb is superior as a reference to [14C]sucrose as the extracellular reference for unchanged [3H]palmitate (Fig. 3A). The difference probably reflects the high binding of palmitate to Alb. Luxon and colleagues (26) used [14C]albumin as their reference for 3,5,3'-triiodothyronine (27) and [14C]sucrose for palmitate. The fitting of unbound and unchanged [3H]palmitate using both slow-diffusion models results in larger MSC compared with other models. However, the slow-diffusion/unbound model was not used in this work due to the additional parameter KR that yielded a higher kinetic parameter standard deviation than in the slow-diffusion/bound model. In addition, it has been shown by Weisiger (41) that the concentration of unbound palmitate in the cytoplasm is orders of magnitude too small to uphold the observed diffusional flux across the cytoplasm. Thus the slow-diffusion/bound model is the only model that both fits the MID data and is consistent with the biophysics of cytoplasmic fatty acid diffusion.

The acinar distribution of L-FABP has been shown to follow the concentration gradient of long-chain fatty acids, i.e., highest in the periportal area and lowest in the perivenous area (4). It is generally accepted that although axial heterogeneity in enzyme activity within the hepatic acinus does not greatly affect the pharmacokinetics of the parent solute (3, 36), it will affect generated metabolite profiles (3).

L-FABP appears to be the major determinant for most of the pharmacokinetic parameters for palmitate. The interrelationships of unchanged palmitate extraction, unchanged palmitate diffusivity (kd) and the L-FABP level (Fig. 4, A-C) emphasizes the role of palmitate diffusion bound to L-FABP as a determinant of its extraction. Uptake and intracellular trafficking of fatty acids has been studied in hepatocyte suspensions (1, 8) and perfused rat liver (17, 18, 26, 39). In those studies, it is generally acknowledged that the transmembrane flux of long-chain fatty acids occurs by diffusion and by a transmembrane transporter (i.e., FABP). Luxon (24) showed that the cytoplasmic transport of 12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazol-amino stearate is modulated by binding to soluble proteins like FABPs and that FABPs enhance diffusive transport by reducing binding to immobile cytosolic membranes. Weisiger and Zucker (42) have recently proposed a simple kinetic model for cytoplasmic diffusion and suggested that "membrane-active" FABPs are the soluble fatty acid binding proteins that enhance faster cytoplasmic diffusion of fatty acids. The correlation between PS for unchanged [3H]palmitate and the L-FABP level (Fig. 5A) suggest that the plasma membrane FABP levels may parallel L-FABP. An analysis of the effects of changing parameters for metabolism (ke), PS, and cytoplasmic diffusion (kd) on steady-state palmitate extraction using the slow-diffusion/bound model showed all parameters effected extraction. Hepatic extraction was most sensitive to changes in metabolic rate followed by permeability. Changes in cytoplasmic diffusion had a lesser effect on the hepatic extraction of palmitate in this work than the other parameters. However, the parameters for metabolic rate and cytoplasmic diffusion may, in reality, be interrelated, increasing the importance of cytoplasmic diffusion as a determinant of extraction.


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Fig. 4.   Relationship between liver FABP (L-FABP) level and diffusion constant, kd (A); kd and hepatic extraction ratio (B); L-FABP level and hepatic extraction ratio of [3H]palmitate in the various animal models (C).



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Fig. 5.   Relationship between L-FABP level and pharmacokinetic parameters derived from the slow-diffusion model fitting for [3H]palmitate in the various animal models permeability surface area product (PS) (A); palmitate metabolic clearance (CLint) (B); production of area-under-the-curve low-molecular-weight metabolites of palmitate (AUCmet) (C); mean transit time, retention of palmitate metabolites in the liver (MTTmet) (D); and retention of palmitate in the liver (kin/kout) (E). , Male; open circle , clofibrate-treated male; black-down-triangle , female; down-triangle, pregnant female.

Retention of palmitate in the liver (kin/kout) was determined by the clearances into and out of the hepatocyte via the sinusoidal membrane and by the distribution spaces for the unbound and unchanged [3H]palmitate between the hepatocyte and the perfusate. One explanation for the increase in the kin/kout ratio for [3H]palmitate (Fig. 5E) is asymmetric transport. A more likely reason is the higher Vc with potential effects on effective mass transport of palmitate. Thus L-FABP regulates the rate of steady removal of fatty acids from plasma by the liver. Such effects may be more pronounced in our studies in which a lower perfusate Alb concentration facilitated higher palmitate extraction than in studies using a higher concentration. Furthermore, the L-FABP level and Vc often correlated.

Clofibrate is one of the peroxisome proliferator-activated receptor alpha  activators, which induce ectopic expression of L-FABP mRNA (higher level) and adipose-FABP mRNA (lower level) in the liver (30). Whether ectopic expression of FABP mRNA results in significant hepatic FABP levels and thus affects the FABP concentrations and long-chain fatty acid pharmacokinetics in the liver is not known and cannot be excluded. However, an investigation of the influence of ectopic FABP levels on long-chain fatty acid hepatic disposition is beyond the scope of this study.

Thus far, no experimental model is available in which, specifically, the concentrations of L-FABP can be manipulated. Such models (for example L-FABP knockout or heterozygous mice) offer an advantage over other models used to study the role of L-FABP in fatty acid hepatic pharmacokinetics.

In conclusion, [3H]palmitate hepatic pharmacokinetics are characterized by an integrated "slow" cytoplasmic diffusion, "instantaneous" binding, barrier limited, and two-phase stochastic distribution model. The parameters hepatic extraction ratio, AUCmet, MTT, and MTTmet derived from moments increased with increasing L-FABP level, whereas CV2 did not differ significantly for any of the animal models studied. The derived pharmacokinetic parameters CLint, PS, kin/kout, and kd for unchanged [3H]palmitate also increased with an increasing L-FABP level in all animal models. Hence, increasing L-FABP levels lead to a greater hepatic transmembrane permeation, diffusion, and palmitate metabolic clearance. Finally, this work paves the way for further exploration of intracellular long-chain fatty acid disposition in intact normal and diseased livers.


    ACKNOWLEDGEMENTS

We thank Michael Weiss for discussions and Young Mo for technical support.


    FOOTNOTES

We acknowledge the support of the National Health and Medical Research Council of Australia and the Queensland and New South Wales Lions Kidney and Medical Research Foundation.

Address for reprint requests and other correspondence: M. S. Roberts, Dept. of Medicine, Univ. of Queensland, Princess Alexandra Hospital, Woollongabba, Qld 4102, Australia (E-mail: M.Roberts{at}mailbox.uq.edu.au).

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. Section 1734 solely to indicate this fact.

First published November 20, 2002;10.1152/ajpgi.00328.2002

Received 6 August 2002; accepted in final form 7 November 2002.


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DISCUSSION
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