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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Uptake of L-Carnitine and Its Short-Chain Ester Propionyl-L-carnitine in the Isolated Perfused Rat Liver

Sigma-Tau SpA, Pomezia, Rome, Italy (A.M., A.L.); Centre for Pharmaceutical Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia (A.M.E.); and Department of Pharmacy Practice, Victorian College of Pharmacy, Monash University, Melbourne, Victoria, Australia (R.L.N.)

Received April 12, 2005; accepted June 9, 2005.

	Abstract

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Hepatic uptake of propionyl-L-carnitine (PLC) and L-carnitine (LC) was assessed with the impulse-response technique in the single-pass perfused rat liver. The experiments involved a rapid injection (impulse) of a mixture of the radiolabeled test compound (PLC or LC) and a reference compound (sucrose) into portal vein inflow and collection and radiochemical analysis (response) of the venous outflowing perfusate samples. The impulse injection was made in the presence of increasing unlabeled background concentrations of PLC (0–50 µM) or LC (50–500 µM) perfusing the liver. The hepatic uptake was minimal or negligible for LC, whereas the hepatic influx clearance was found to be low (0.095 ml/s equivalent to 5.7 ml/min) for PLC relative to the perfusate flow rate (30 ml/min). When background concentrations of PLC were increased (from 1–50 µM), the influx clearance was reduced in a concentration-dependent behavior, indicating partial saturation of the entry of compound into hepatocytes. PLC was taken up into hepatocytes via a unidirectional transport process with negligible efflux. The hepatic uptake of PLC was significantly reduced in the presence of unlabeled LC (500 µM), indicating an inhibition of the sinusoidal membrane transport of PLC by LC. The study showed the sinusoidal membrane is a permeability barrier to the entry of PLC and LC into hepatocytes, and it is the site of a common carrier-mediated transporter for both compounds.

Pharmacokinetic studies in humans involving oral and intravenous administration of LC have shown an absolute oral bioavailability of less than 20% (Harper et al., 1988; Sahajwalla et al., 1995; Evans and Fornasini, 2003). Although there are no published data on the oral bioavailability of PLC, data from studies in both humans and experimental animals suggest a similar bioavailability (<20%) for this compound (A. Longo, personal communication). Both LC and PLC are highly polar compounds, possessing both cationic and anionic functional groups at physiological pH, and move across biological membranes via carrier-mediated transport systems (Lahjouji et al., 2001). The observed limited bioavailability of LC and PLC may be because of poor absorption and/or extensive presystemic first-pass biotransformation.

Recently, using a perfused rat liver preparation, we have shown hepatic extraction ratios of 0.022 and 0.115 for LC and PLC, respectively (Mancinelli et al., 2000). Considering the low hepatic extraction ratio values, it is unlikely that the liver contributes to the low bioavailability of these compounds in humans and experimental animals (Harper et al., 1988; Sahajwalla et al., 1995; Evans and Fornasini, 2003). However, our earlier study (Mancinelli et al., 2000) did not identify whether the low hepatic extraction ratios for LC and PLC are the result of hepatic uptake-rate limitation (transport-rate limitation) or of a low intrinsic clearance for hepatic metabolism (metabolic-rate limitation).

The aim of the present study was to determine whether the uptake of LC and PLC is a transport rate-limited process and if so, whether this process is the rate-limiting factor in the overall extraction of these compounds by the liver. The study was also designed to investigate the hypothesis that LC would inhibit the hepatic uptake of PLC and thereby reduce its extraction ratio. The aims were addressed using the impulse-response (IR) technique in the isolated perfused rat liver (Evans et al., 1993). A rapid injection (impulse) of labeled test compound (LC or PLC), along with the appropriate reference marker, was made against a range of background concentrations of unlabeled LC or PLC. Because both LC and PLC are nonprotein-bound and low molecular weight substances (Marzo et al., 1991), the reference compound was labeled sucrose, a frequently used marker of the combined vascular space and the space of Disse (Goresky and Nadeau, 1974).

	Materials and Methods

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Chemicals. [¹⁴C]PLC, [³H]LC, [³H]sucrose, and [¹⁴C]sucrose were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). The specific activities were 0.056, 79, 0.0132, and 0.615 Ci/mmol, respectively. The radiochemical purity for all the radiolabeled compounds was greater than 98%, as assessed using high-performance liquid chromatography (HPLC). Unlabeled PLC and LC compounds were obtained from Sigma-Tau (Pomezia, Rome, Italy). The liquid scintillation fluid (ACS) was purchased from GE Healthcare. Other reagents were of analytical grade or equivalent and were purchased commercially.

Animals. Male Sprague-Dawley rats (270–360 g) were supplied by Gilles Plains Animals Resource Center (Adelaide, South Australia). The animals were kept in a temperature-controlled room (22 ± 2°C), 50 to 60% relative humidity under a 12-h light/dark cycle with free access to water and food. The institutional animal ethics committee approved the study.

Rat Isolated Perfused Liver Preparation. The in situ perfused rat liver preparation was based on previously described methods (Evans and Shanahan, 1995; Mancinelli et al., 2000). Briefly, animals were anesthetized with sodium pentobarbital (60 mg/kg, Nembutal; Boehringer Ingelheim, NSW, Australia), the liver was exposed via a midline incision, and the common bile duct was cannulated with polyethylene tubing (PE 10; Paton Scientific, Victor Harbor, South Australia). Bile was collected into preweighed tubes, and the bile flow rate was determined gravimetrically assuming a specific gravity of 1. The portal vein was cannulated with an 18-gauge catheter (BD Biosciences, Franklin Lakes, NJ), and the liver was perfused with an albumin- and erythrocyte-free Krebs'-bicarbonate buffer solution, pH 7.4. This solution was prepared on the day of perfusion, filtered through a 0.2-µm membrane (Millipore Corporation, Billerica, MA), supplemented with D-glucose (15 mM) and sodium taurocholate (8.33 mM), and saturated with 95% O₂/5% CO₂. The perfusate was delivered to the hepatic portal vein cannula at a constant flow rate of 15 ml/min via a Masterflex model 7518-00 peristaltic pump (Cole-Parmer Instrument Co., Vernon Hills, IL). The liver was perfused in a single-pass fashion in which a 14-gauge intravenous cannula (BD Biosciences), with a draining tube attached, was inserted through the right atrium of the heart into the vena cava for the collection of hepatic perfusate effluent. Finally, the suprarenal inferior vena cava was ligated, resulting in the complete vascular isolation of the liver and unidirectional flow of perfusate through the organ. The perfusate flow rate was then increased to 30 ml/min, and the animal was placed in a temperature-controlled perfusion cabinet at 37°C. The exposed liver was moistened with saline and covered with a piece of parafilm. Liver viability was assessed by macroscopic appearance, bile production, oxygen consumption, and percentage recovery of perfusate (Mancinelli et al., 2000).

IR Experiment. This methodology essentially follows that for the multiple indicator dilution studies as used in previous studies (Wolkoff et al., 1987; Chou et al., 1993; Evans et al., 1993). After a 15- to 20-min stabilization period, the perfusate was changed through a three-way valve (Discofix; B. Braun, Melsungen, Germany) to one containing a background concentration of unlabeled drug (see under Experimental Design) to enter the liver. After 5 min, 25 µl of perfusate solution containing 0.05 µCi of [³H]sucrose (reference compound) and 0.0625 µCi of [¹⁴C]PLC (test compound) for PLC experiments, or 0.1 µCi of [¹⁴C]sucrose (reference compound) and 0.5 µCi [³H]LC (test compound) for LC experiments, was rapidly injected as a bolus (impulse) via a Y-piece device placed immediately proximal to the portal vein cannula without disruption of perfusate supply. The length of tubing between the injection site and the portal vein cannula and that between the venous cannula and the collection apparatus were kept to a minimum. Immediately after an injection, the total hepatic effluent was collected automatically for 60 s with a purpose-built motor-driven carousel with 60 sampling holes, at successive 1.0-s intervals for 30 s. The total effluent was also sampled manually at 1.5, 2, 2.5, 3, 4, 5, and 9 min. The perfusate was then changed to one containing a different unlabeled concentration of LC or PLC (see under Experimental Design). After a further 5 min, another injection was made, and the samples were collected as before. Third and fourth tracer 25-µl injections over a different background concentration of unlabeled drug were also performed.

Experimental Design. To investigate the effect of concentration on the hepatic transport kinetics of LC and PLC, two sets of experiments were performed. In the first set of experiments (n = 3), each liver was sequentially perfused with four different background concentrations of unlabeled LC: period 1, 50 µM; period 2, 100 µM; period 3, 200 µM; and period 4, 500 µM. For each period, a 25-µl bolus (impulse) containing reference ([¹⁴C]sucrose) and test compound ([³H]LC) was injected (see under IR Experiment). Each period was no longer than 15 min. In the second set of experiments (n = 4), each liver was sequentially perfused with four different background concentrations of unlabeled PLC: period 1, 0.0 µM; period 2, 1.0 µM; period 3, 5.0 µM; and period 4, 50 µM. For each period, a 25-µl bolus (impulse) containing reference ([³H]sucrose) and test compound ([¹⁴C]PLC) was injected. Each period was no longer than 15 min.

To investigate the effect of LC input concentrations on the hepatic transport kinetics of PLC, a third set of experiments was performed. Each liver (n = 4) was sequentially perfused with four different background concentrations of unlabeled LC: period 1, 0.0 µM; period 2, 50 µM; period 3, 200 µM; and period 4, 500 µM. For each period, a 25-µl bolus (impulse) containing reference ([³H]sucrose) and test compound ([¹⁴C]PLC) was injected.

Two additional studies were performed to identify whether changes in the hepatic disposition of PLC, apparently caused by increasing perfusate concentrations of PLC and LC, could also be explained by changes with time in the performance of the isolated perfused liver preparation. For the first of these experiments, each liver (n = 2) was perfused with drug-free perfusate for periods 1 through 4. In the second study, each liver (n = 2) was perfused with sequential unlabeled PLC concentrations in the perfusate as follows: period 1, 0.0 µM; period 2, 50 µM; period 3, 5.0 µM; and period 4, 1.0 µM. For both of the above-mentioned studies, a 25-µl bolus containing reference ([³H]sucrose) and test compound ([¹⁴C]PLC) was injected.

Radiochemical Analysis. All the outflowing perfusate and bile samples were analyzed for [³H] and [¹⁴C] radioactivity on a Tri-Carb 2200CA liquid scintillation counter (PerkinElmer Life and Analytical Sciences, Boston, MA). This enabled the simultaneous determination of the [¹⁴C]PLC or [³H]LC and the ³H- or ¹⁴C-labeled sucrose. For each perfusate sample, a 0.2- to 0.4-ml aliquot was mixed with 4.5 ml of ACS liquid scintillant fluid. After mixing, each vial was kept in darkness for 14 to 16 h, after which radioactivity was counted for 5 min, with counts per minute values converted to disintegrations per minute values using [³H] and [¹⁴C] quench curves. For bile, a 50-µl aliquot was mixed with 0.4 ml of deionized water, and 4.5 ml of ACS liquid scintillant fluid was added. Radioactivity was measured as for the perfusate samples. To measure the true dose of the administered [³H] and [¹⁴C] tracer compounds, a 25-µl aliquot of bolus (impulse) was mixed with 0.2 to 0.4 ml of ACS liquid scintillant fluid. Radioactivity was measured as described above. Because our recent studies showed that the rat liver is capable of converting PLC to LC and also LC to its short-chain ester acetyl-L-carnitine, possible metabolite formation during the IR experiments was therefore examined using an HPLC method as previously described (Mancinelli et al., 2000).

Data Analysis. The determination of hepatic kinetic parameters followed methods described previously (Kakutani et al., 1985; Evans et al., 1993). The frequency output (F_out) of each tracer compound in effluent perfusate, at each sampling time, was calculated from the output concentration (dpm/ml), by the following transformation:

(1)

where Q is the perfusate flow rate through the liver, and the units of F_out are per second. Expressed in this manner, F_out represents the fraction of the dose eluting per second. The area under the frequency output versus sampling time profile from time 0 to infinity (AUC), determined by the linear trapezoidal rule with extrapolation to infinite time, gives an estimate of the total fractional recovery. In each case, the extrapolated area was taken to be the F_out at the final time point (30 s), divided by the slope determined by log-linear regression of the terminal portion (last five F_out values; 25–30 s) of the profile. Hepatic availability (F_H) was calculated from the ratio of the AUC of the test substance (AUC_test) to that of sucrose, the vascular reference compound, (AUC_sucr) as follows:

(2)

The hepatic extraction ratio (E_H) was calculated as:

(3)

Equation 2 is based on the assumption that the extracellular reference compound (sucrose) was not extracted by the liver and that its recovery from effluent perfusate was 100%. Experimental observations proved this assumption to be valid. The mean transit time (MTT) of tracer was calculated by moment analysis as follows:

(4)

where AUMC is the area under the first moment curve from zero to infinity, determined by the linear trapezoidal rule as described above with extrapolation to infinite time. For each MTT of labeled compound (reference or test), the transit time through the injection device and the collection catheter was subtracted (in these experiments the value was 4.4 s) to estimate the mean residence time (MRT) through the liver as follows:

(5)

The ratio of the F_out of the reference (sucrose) to that of the test substance, expressed as the natural logarithm, was plotted versus time. The initial slope of this plot (from 6–16 s in the current experiments), calculated by linear regression, reflected the hepatic influx rate constant (K_in) for the test substance (Ishigami et al., 1995). The hepatic influx clearance of test substance () was calculated, using the following equation:

(6)

where V_sucr was the volume accessible to the extracellular reference compound sucrose. The hepatic distribution volume V, for reference and test compounds, was determined as:

(7)

Data are expressed as mean ± S.D. A repeated-measures one-way analysis of variance, followed by a two-tailed Dunnett's test, was used to compare the hepatic kinetic parameters from periods 2 through 4 with the initial value (period 1). An unpaired Student's t test was used to assess differences in period 1 values between PLC concentration-ranging experiments and PLC-LC interaction experiments. A p value less than 0.05 was considered statistically significant. In the concentration-ranging study with PLC, a correlation analysis was used for the determination of the statistical significance of the relationship between E_H and K_in. The analysis was performed by the use of individual period values (periods 1–4) for E_H and K_in at each concentration of PLC.

	Results

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Radioactivity in Bile Samples and Evaluation of Perfusate Samples by Chromatography. At the end of each experiment, the total radioactivity in bile collected over the experimental period was determined, and no radioactivity was observed. This suggests no excretion of the injected labeled compounds into the bile. For both LC and PLC experiments, the only identified radiolabeled compound detected in outflowing perfusate on HPLC analysis was that of the compound injected ([³H]LC or [¹⁴C]PLC). Therefore, in all the cases, no metabolites were detected in the effluent samples, indicating no interference with the evaluation of total count (dpm/ml) of [³H]LC or [¹⁴C]PLC in IR experiments.

Concentration-Ranging Study with LC. A set of representative frequency output versus time profiles for [³H]LC and [¹⁴C]sucrose at four background concentrations of unlabeled LC (50, 100, 200, and 500 µM) is shown in a semilogarithmic format in Fig. 1. The size and shape of the output profiles of [³H]LC were similar to those of the reference compound [¹⁴C]sucrose and were not sensitive to changes in background concentrations of unlabeled LC. Because the curves of [³H]LC and [¹⁴C]sucrose overlapped extensively during the early phase of the output profiles, K_in values, which were very close to zero at all four background concentrations of unlabeled LC, could not be determined with confidence. This behavior suggests minimal or no uptake of LC into the liver. This was confirmed by hepatic availability for [³H]LC of 101 ± 1% at all the LC concentrations examined. However, careful inspection of the terminal phase (from 15–30 s) shows the possibility of a very minor, delayed efflux of LC (Fig. 1).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Semilogarithmic plot of Fout versus sampling time for [3H]LC (closed squares) and [14C]sucrose (open circles) with unlabeled LC background concentrations of 50 µM (A), 100 µM (B), 200 µM (C), and 500 µM (D) of LC in perfusate in a representative IR experiment.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Semilogarithmic plot of Fout versus sampling time for [14C]PLC (closed squares) and [3H]sucrose (open circles) with unlabeled PLC background concentrations of 0 µM (A), 1.0 µM (B), 5.0 µM (C), and 50.0 µM (D) of PLC in perfusate in a representative IR experiment.

A plot of the natural logarithm of the ratio of F_out of reference compound (sucrose) to that of test compound (PLC) as a function of time, in the absence of background PLC concentrations in perfusate, is shown in Fig. 3, with the log-ratio increasing linearly with time (Fig. 3).

View larger version (9K): [in this window] [in a new window]   Fig. 3. Natural logarithm of the ratio of Fout of [3H]sucrose to that of [14C]PLC versus time in the absence of unlabeled PLC in perfusate in IR experiments (p < 0.001; r2 = 0.992). Each point and vertical bar represent the mean ± S.D. of four different experiments.

The K_in and values of [C]PLC decreased progressively as the PLC concentrations increased, and the values observed at 50 µM PLC were significantly lower (p < 0.01) than those observed with no background PLC present in perfusate (Table 1). A highly significant (p < 0.001) positive relationship between the E_H and K_in of PLC was observed as shown in Fig. 4. Interestingly, radiometric analysis of outflow perfusate samples, taken from 60 s to 9 min for each period (periods 1–4) of IR experiments, did not reveal any radioactivity of PLC. This finding suggests PLC, which was taken up into hepatocytes, did not reappear later in the outflowing samples (data not shown).

View larger version (8K): [in this window] [in a new window]   Fig. 4. Relationship between extraction ratio and influx rate constant (s–1) of [14C]PLC in concentration-ranging study with PLC (p < 0.001; r2 = 0.637).

Reproducibility and Reversed Concentration-Ranging Studies. The viability and reproducibility of the perfused liver preparation were assessed in two livers by performing four period IR experiments with [¹⁴C]PLC in the absence of PLC in perfusate. The hepatic extraction ratio of PLC and the kinetic parameters remained constant across the four periods (E_H, 0.130 ± 0.013; K_in, 0.042 ± 0.005 s^–1; , 0.097 ± 0.013 ml/s), indicating that the viability and the reproducibility of the preparation was not affected by perfusion time.

In addition, two experiments with an initial period with no PLC in perfusate, followed by periods 2 through 4 with decreasing concentrations of unlabeled PLC (from 50 µMto1.0 µM), were carried out. With no background PLC in the perfusate, the E_H (0.099), K_in (0.043 s^–1), and (0.102 ml/s) values of PLC were similar to those obtained under identical conditions with the PLC concentration-ranging study (Table 1). When the concentration of unlabeled PLC in perfusate was increased to 50 µM in period 2, the kinetic parameters decreased (E, 0.072; K_in, 0.028 s^–1; , 0.063 ml/s) to values that were close to those for the PLC concentration-ranging study, in which the concentration of unlabeled PLC in perfusate was 50 µM in period 4 (Table 1).

	Discussion

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Using a recirculating isolated perfused liver system, the hepatic disposition of LC and PLC was previously investigated (Mancinelli et al., 2000). In that study, LC or PLC was introduced into a reservoir that was repeatedly sampled for 90 min to assess pharmacokinetic parameters, such as half-life, hepatic clearance, and hepatic extraction ratio (Mancinelli et al., 2000). Although the E_H of PLC (0.115) was 5-fold that of LC (0.022), the values for both compounds indicated that their elimination is limited not by the hepatic perfusion rate but by the low capacity of liver to remove them (Mancinelli et al., 2000). This low capacity could arise from either low activity of the transport systems involved in the movement of these polar substrates into the intracellular hepatic environment or the limited capacity of the hepatic intracellular enzymes to mediate their metabolism. In the present study, data from IR experiments provide evidence that the sinusoidal membrane is a permeability barrier to the entry of these two compounds into hepatocytes. However, the observed behavior of LC in the liver is quite different compared with that of PLC.

When tracer LC was injected into the portal vein in the presence of a background unlabeled LC concentration encountered physiologically in rat and human plasma (Longo et al., 1996; Pessotto et al., 1996), the availability of LC was close to 100%. This value also remains constant when the background concentration of LC increased 10-fold. As a consequence, the E_H was very low, and this is in agreement with the results of the study in which isolated perfused rat livers were perfused with 50 µM of LC in a recirculating mode (Mancinelli et al., 2000). In this latter study, the extraction ratio value of LC was reported to be 0.022 ± 0.015. In the present IR study, the output curves of [³H]LC and [¹⁴C]sucrose were almost superimposable, suggesting that a major proportion of the LC activity passed through the liver as a "throughput" component, without entering liver cells. However, a slight divergence in shape between the [³H]LC and [¹⁴C]sucrose curves at later time points (Fig. 1) suggests that a very small component of LC had entered hepatocytes and subsequently returned to the circulation. In keeping with these results, the inhibitory effect of LC on the uptake of PLC (see below) also suggests that some uptake of LC at the sinusoidal level occurs (Table 2).

The relationship between the [¹⁴C]PLC and [³H]sucrose outflow curves provides the information needed to account for what is happening to PLC in the absence of background material (Fig. 2). The extraction ratio for PLC under these conditions, estimated in a model-independent manner, confirms the uptake of [¹⁴C]PLC into the liver. This is in agreement with the previous studies in the recirculating isolated perfused rat liver (Mancinelli et al., 2000) and with in vivo studies in rats (Davenport et al., 1995). In addition, the value of being substantially less than perfusate flow rate (5.7 versus 30 ml/min; Table 1) suggests that uptake of PLC into the liver is a permeability-limited process.

The traditional multiple indicator dilution model of Goresky et al. (1993) considers the liver as an array of parallel channels of varying length, with each channel surrounded by an extracellular-extravascular (Disse) space and a plate of liver cells. In this model, the transfer of dissolved substances in the perfusate crossing into the liver is governed by the k_in, k_out, and k_seq parameters, which correspond to the influx, efflux, and sequestration rate constants, respectively (Goresky et al., 1993). During IR experiments with PLC, the influx rate constant k_in was well defined by the data, whereas the estimates of k_out and k_seq could not be made. With the k_out and k_seq rate constants set to zero, the flow-limited distribution model of Goresky simplifies to the one-parameter model, which is more simplistic. Interestingly, the inspection of the late tailing component of the [¹⁴C]PLC curve (Fig. 2A) showed no evidence of substantial late return of [¹⁴C]PLC into the perfusate. If return of [¹⁴C]PLC to perfusate occurs, it would have been expected to lead to a downward deviation of the log ratio versus time curve later in time (Goresky and Nadeau, 1974). This behavior was not observed for PLC, which presents an upward straight line constant for the first 16 s (p < 0.001; r² = 0.992; Fig. 3).

The low values of E_H for PLC in these IR experiments at low background concentrations were close to those previously reported in the recirculating isolated rat liver preparation when the initial perfusate concentration was 0.45 µM (Mancinelli et al., 2000). However, a significant (p < 0.05) decrease in the K_in, , and E_H values was observed at a perfusate PLC concentration of 50 µM. This finding suggests a concentration-dependence in the uptake process of PLC into the liver.

It could be argued that the concentration-related decrease in K_in, , and E_H across periods 1 to 4 may be caused by decreasing viability of the liver preparation over the experimental period or by an accumulating effect of increasing background concentration of PLC through periods 2 to 4. These possibilities are easily rejected as shown by the good viability of the preparation as evidenced by a lack of change, with time, in the pivotal kinetic parameters. Moreover, the values of K (0.028 s^–1), (0.063 ml/s), and E_H (0.072) of PLC are comparable with those encountered in Table 1, although the background concentration of PLC decreased from 50 µM in period 2 to 1 µM in period 4.

Previous in vitro studies showed that LC is transported actively into hepatocytes and that compounds with closely related chemical structures, such as the direct precursor of LC, -butyrobetaine, share the same uptake transport system (Christiansen and Bremer, 1976; Nakajima et al., 1999; Yokogawa et al., 1999). Because it is likely that LC also shares the same transport system with short-chain carnitine esters in other isolated perfused organs (Mancinelli et al., 1995; Evans et al., 1997), the IR technique was used to investigate whether the hepatic kinetic parameters of [¹⁴C]PLC were affected by the presence of increasing concentrations of LC. At physiological plasma concentrations of LC, the hepatic kinetic parameters of PLC were not altered. However, once the concentrations of LC were 4 to 10 times higher than those encountered physiologically, the K_in and values for PLC decreased significantly (p < 0.05). This observation shows that LC inhibited PLC influx in a concentration-dependent manner and that LC and PLC could share a common influx transport system.

The observed saturable component of PLC liver uptake in our IR experiments taken together with the competitive inhibition by LC on PLC uptake are entirely consistent with a carrier-mediated transport for the entry of PLC (and LC) into the liver. In fact, a large number of studies have reported that a family of organic transporters designated organic/carnitine transporter (Octn) performs LC transport in cell membranes (Tamai et al., 1997; Koepsell, 2004). Some novel members of this family, namely, Octn1, Octn2, and Octn3, have been identified and showed ability to transport LC in animal and human cell membranes (Lahjouji et al., 2001). In both rat and human species, Octn2, likely the most important LC transporter, was also able to transport the short-chain acyl esters of LC such as acetyl-L-carnitine and PLC in cells expressing this transporter (Wu et al., 1999). Interestingly, the authors have shown that LC inhibited the Octn2-mediated transport for acetyl-L-carnitine and PLC in agreement with the results of LC inhibition on PLC hepatic uptake in the present IR experiments.

In general, Octn2 exhibited the greatest expression in the kidney, where it is mainly involved in carnitine reabsorption (Tamai et al., 2000). In contrast, a low level (but detectable) expression of Octn2 has been reported in the liver (Tamai et al., 1998; Slitt et al., 2002; Choudhuri et al., 2003). The low expression of Octn2 in the liver could explain the low E_H of both LC and PLC observed during the perfusion of these compounds in both the present and previous studies (Mancinelli et al., 2000). Further studies could explore the physiological (and pharmacological) significance of Octn2 on the hepatic sinusoidal uptake of LC and its short-chain acyl ester PLC.

In conclusion, the results of this investigation showed that for LC the entry into hepatocytes is very low. For PLC, the E_H, K_in, and are low, and its influx into the liver cell is concentration-dependent. In addition, this influx is inhibited by the presence of LC. Thus, the sinusoidal membrane is a permeability barrier to entry of PLC and LC into hepatocytes.

	Footnotes

 
This work was financially supported by Sigma-Tau SpA, Pomezia, Rome, Italy.

doi:10.1124/jpet.105.087890.

ABBREVIATIONS: LC, L-carnitine; PLC, propionyl-L-carnitine; IR, impulse response; HPLC, high-performance liquid chromatography; ACS, aqueous counting scintillant; AUC, area under the curve; MTT, mean transit time; Octn, organic/carnitine transporter.

Address correspondence to: Angelo Mancinelli, Sigma-Tau SpA, Via Pontina Km 30,400, Pomezia (Rome), Italy, 00040. E-mail: angelo.mancinelli{at}sigma-tau.it

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