uptake of l-carnitine and its short-chain ester propionyl...

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Uptake of L-carnitine and its Short-chain Ester Propionyl-L-carnitine in the Isolated Perfused Rat Liver

Angelo Mancinelli, Allan M. Evans, Roger L. Nation and Antonio Longo

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.)

JPET Fast Forward. Published on June 10, 2005 as DOI:10.1124/jpet.105.087890

Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 10, 2005 as DOI: 10.1124/jpet.105.087890

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Running Title

Hepatic Uptake of Carnitines

Corresponding Author Angelo Mancinelli, M. Pharm. Sigma-Tau SpA Via Pontina Km 30,400 Pomezia (Rome), Italy, 00040 Phone: + 39 (06) 91393656 Facsimile: + 39 (06) 91393940 e-mail: [email protected] Number of text pages (including abstract & references) 24 Number of references 39 Number of tables 2 Number of figures 4 Number of words in the Abstract 232 Number of words in the Introduction 608 Number of words in Discussion 1427

Non-standard abbreviations ANOVA, analysis of variance; AUC, area under the curve; AUMC, area under the first

moment curve; CLHin , hepatic influx clearance; [14C]PLC, propionyl-L-[N-methyl-

14C]carnitine HCl; CPM, count per minute; [14C]sucrose, [U-14C]-sucrose, DPM, disintegration per minute; EH, hepatic extraction ratio; FH, hepatic availability; Fout,

frequency output; [3H]LC, L-[N-methyl-3H]carnitine; [3H]sucrose, [6,6’(n-)-3H]-sucrose; IR, impulse response; Kin, hepatic influx rate constant; kin, influx rate constant; kout, efflux rate

constant; kseq, sequestration rate constant; LC, L-carnitine; MRT, mean residence time; MTT, mean transit time; PLC, propionyl-L-carnitine; Q, perfusate flow rate; V, volume of distribution of test or reference compound; Vsucr volume accessible to sucrose, the interstitial

reference compound. Recommended section assignment Absorption, Distribution, Metabolism, & Excretion


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Abstract Hepatic uptake of propionyl- (PLC) and L-carnitine (LC) was assessed with the

impulse response (IR) technique in the single-pass perfused rat liver. The experiments

involved a rapid injection (impulse) of a mixture of the radiolabeled test compound

(propionyl- or L-carnitine) 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. For LC the hepatic

uptake was minimal or negligible, whereas for PLC the hepatic influx clearance was found to

be low (0.095 ml • sec-1 equivalent to 5.7 ml • min-1) relative to the perfusate flow rate (30 ml

• min-1). When background concentrations of PLC were increased (from 1 to 50 µM) the

influx clearance was reduced in a concentration-dependent behaviour, 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

significally reduced in the presence of unlabeled LC (500 µM) indicating an inhibition of the

sinusoidal membrane transport of PLC by LC. The study demonstrated 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.


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Introduction

LC is an endogenous compound that has important physiological roles, including

involvement in the β-oxidation of fatty acids by facilitating the transport of long chain fatty

acids across the inner mitochondrial membrane (Bremer, 1983; Bahl and Bressler, 1987). In

mammals, the body stores of LC are maintained by a combination of absorption from dietary

sources, endogenous biosynthesis by numerous organs (primarily in the liver), and highly

efficient reabsorption in the kidney (Bremer, 1983; Rebouche and Seim, 1998; Hoppel and

Davis 1986; Evans and Fornasini 2003). Carnitine supplementation has been used

therapeutically for treatment of primary and secondary carnitine deficiency, caused by a

defective carnitine transport in the plasma membrane (Nezu et al., 1999) or associated with

genetically determined metabolic errors, acquired medical conditions or iatrogenic states

(Pons and DiMauro, 1998). Early evidence suggested that the compound may be used

beneficially for the treatment of cardiovascular conditions such as ischemic heart disease and

angina pectoris (Bahl and Bressler, 1987; Goa and Brogden, 1987). A number of short-chain

carnitine esters of LC have been proposed for pharmacological use. For example, PLC which

is produced by esterification of the hydroxyl group of LC and it is an important component of

the endogenous carnitine pool, has been evaluated for the treatment of peripheral arterial

diseases and other cardiovascular disorders (Böhmer and Bremer, 1968; Brevetti et al. 1992;

Hiatt, 2001; Duprez et al. 2003; Bartels et al., 1992).

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


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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 due to poor absorption and/or extensive presystemic first-pass biotransformation.

Recently, using a perfused rat liver preparation, we have demonstrated 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 due to

hepatic uptake-rate limitation (transport-rate limitation) or to 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 limitated 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. Since both LC and PLC are

non-protein-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).


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Materials and Methods Chemicals. [14C]PLC, [3H]LC, [3H]sucrose and [14C]sucrose were purchased from Amersham

Pharmacia Biotech (Buckinghamshire, UK). The specific activities were 0.056, 79, 0.0132,

and 0.615 Ci/mmol, respectively. The radiochemical purity for all radiolabeled compounds

was greater than 98%, as assessed using HPLC. Unlabeled PLC and LC compounds were

obtained from Sigma Tau (Pomezia, Rome, Italy). The liquid scintillation fluid (ACS) was

purchased from Amersham (Buckinghamshire, UK). Other reagents were of the 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), 50-60% 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 anaesthetised with sodium pentobarbital (60 mg/kg, Nembutal,

Boehringer Ingelheim, NSW, Australia), the liver was exposed via a mid-line incision, and

the common bile duct was cannulated with polyethylene tubing (PE 10, Paton Scientific,

Victor Harbor, South Australia). Bile was collected into pre-weighed tubes and the bile flow

rate was determined gravimetrically assuming a specific gravity of 1. The portal vein was

cannulated with a 18-gauge catheter (Becton Dickinson, Sandy, UT), and the liver was

perfused with an albumin- and erythrocyte-free Kreb’s-bicarbonate buffer (pH 7.4) solution.

This solution was prepared on the day of perfusion, filtered through a 0.2-µm membrane

(Millipore Corp.; Bedford, 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


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the hepatic portal vein cannula at a constant flow rate of 15 ml • min-1 via a Masterflex model

7518-00 peristaltic pump (Cole Palmer, Vermon Hill, IL). The liver was perfused in a single-

pass fashion in which a 14-gauge intravenous cannula (Becton Dickinson), 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-1 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).

Impulse-Response Experiment. This methodology essentially follows that for the multiple

indicator dilution studies as employed in previous studies (Wolkoff et al., 1987; Evans et al.,

1993: Chou et al., 1993). After a 15- to 20-min stabilisation 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 Experimental Design) to enter the liver.

After 5 min, 25µl 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 sec with a purpose-built motor-driven carousel


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with 60 sampling holes, at successive 1.0 sec intervals for 30 sec. The total effluent was also

sampled manually at 1.5, 2, 2.5, 3, 4, 5, 9 min. The perfusate was then changed to one

containing a different unlabeled concentration of LC or PLC (see Experimental Design). After

a further 5 min, another injection was made, and 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 Impulse-Response Experiment).

Each Period entailed no more than 15 min duration. 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 entailed no more than 15 min duration.

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 due to increasing perfusate concentrations of PLC and LC,


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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 1-4. 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 studies, a 25 µl bolus

containing reference ([3H]sucrose) and test compound ([14C]PLC) was injected.

Radiochemical Analysis. All outflowing perfusate and bile samples were analysed for [3H]

and [14C] radioactivity on a Tri-Carb 2200CA liquid scintillation counter (Packard Instrument,

Victoria, Australia). This enabled the simultaneous determination of the [14C]PLC or [3H]LC

and the 3H- or 14C-labeled sucrose. For each perfusate sample, a 0.2-0.4 ml aliquot was mixed

with 4.5 ml of ACS liquid scintillant fluid. After mixing, each vial was kept in darkness for

14-16 hr after which radioactivity was counted for 5 min, with CPM values converted to DPM

using [3H] and [14C] quench curves. For bile, a 50 µl aliquot was mixed with 0.4 ml of

deionised 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 0.2-0.4 ml of ACS

liquid scintillant fluid. Radioactivity was measured as described above.

Since in our recent studies we demonstrated 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 impulse-response experiments was therefore examined using a high

performance liquid chromatography method as previously described (Mancinelli et al., 2000).

Data Analysis. The determination of hepatic kinetic parameters followed methods described

previously (Evans et al., 1993; Kakutani et al., 1985). The frequency output (Fout) of each


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tracer compound in effluent perfusate, at each sampling time, was calculated from the output

concentration (DPM / ml), by the following transformation:

Fout = (DPM / ml of perfusate) x Q

DPM of Dose (1)

where Q is the perfusate flow rate through the liver, and the units of Fout are sec-1. Expressed

in this manner, Fout represents the fraction of the dose eluting per second. The area under the

frequency output versus sampling time profile from time zero 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 Fout at the final

time point (30 sec), divided by the slope determined by log-linear regression of the terminal

portion (last 5 Fout values; 25-30sec) of the profile. Hepatic availability (FH) was calculated

from the ratio of the AUC of the test substance (AUCtest) to that of sucrose, the vascular

reference compound (AUCsucr) as follows:

FH = AUC

AUCtest

sucr (2)

The hepatic extraction ratio (EH) was calculated as:

EH = 1 - FH (3)

Equation (2) is based on the assumption that the extracellular reference compound (sucrose)

was not extracted by the liver, and 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:

MTT = AUMC

AUC (4)


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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 sec) to

estimate the mean residence time through the liver (MRT) as follows:

MRT = MTT - 4.4sec (5)

The ratio of the Fout 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 to 16 sec in

the current experiments), calculated by linear regression, reflected the hepatic influx rate

constant (Kin) for the test substance (Ishigami et al., 1995). The hepatic influx clearance of

test substance (CLHin ) was calculated, using the following equation:

CLHin = Kin x Vsucr (6)

where Vsucr was the volume accessible to the extracellular reference compound sucrose. The

hepatic distribution volume V, for reference and test compounds, was determined as:

V = Q x MRT (7)

Data are expressed as mean ± S.D. A repeated measures one-way analysis of variance

(ANOVA), followed by a two-tailed Dunnett's test, was used to compare the hepatic kinetic

parameters from Periods 2-4 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 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 EH and Kin. The


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analysis was performed by the use of individual Period values (Periods 1-4) for EH and Kin at

each concentration of PLC.

Results

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 upon HPLC analysis was

that of the compound injected ([3H]LC or [14C]PLC). Therefore, in all 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 impulse-response experiments.

Concentration-ranging study with L-carnitine

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) are shown in

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. Since the curves of [3H]LC and [14C]sucrose

overlapped extensively during the early phase of the output profiles, Kin values, which were

very close to zero at all four background concentrations of unlabeled LC, could not be

determined with confidence. This behaviour suggests minimal or no uptake of LC into the

liver. This was confirmed by a hepatic availability for [3H]LC of 101 ± 1% at all LC

concentrations examined. However, careful inspection of the terminal phase (from 15 to

30sec) shows the possibility of a very minor, delayed efflux of LC (Fig.1).

Concentration-ranging study with propionyl-L-carnitine


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A set of representative frequency output versus time profiles for [14C]PLC and

[3H]sucrose with increasing background concentrations of unlabeled PLC (0-50 µM) are

shown in semilogarithmic format in Fig. 2. The difference between the areas under

[3H]sucrose and [14C]PLC curves provides an index of [14C]PLC extraction. With no

background PLC in the perfusate, the EH of [14C]PLC was 0.141 ± 0.029 (Table 1). The EH

value decreased significantly as the concentrations of unlabeled PLC in the perfusate

increased from 1.0 µM to 50 µM, with a dramatic reduction at 50 µM (p< 0.01; Table 1)

indicating a concentration-dependent mechanism in the hepatic extraction of PLC.

A plot of the natural logarithm of the ratio of Fout 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).

The Kin and CLHin values of [14C]PLC decreased progressively as the PLC concentrations

increased and the values observed at 50 µM PLC were significally 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 EH and Kin of PLC was observed as shown in Fig. 4.

Interestingly, radiometric analysis of outflow perfusate samples, taken from 60 sec. to 9 min

for each Period (Periods 1-4) of IR experiments, did not reveal any radioactivity of PLC. This

finding suggests propionyl-L-carnitine, which was taken up into hepatocytes, did not reappear

later in the outflowing samples (data not shown).

Effect of LC on hepatic kinetic parameters of propionyl-L-carnitine

To determine the influence of LC on the hepatic kinetic parameters of [14C]PLC, livers

were perfused with increasing LC concentrations (from 0 to 500 µM). At each concentration


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of LC (0, 50, 200 and 500 µM), a [14C]PLC impulse-response was conducted (see

Experimental Design). The results are shown in Table 2. Although no statistically significant

decrease in EH for PLC occurred as the perfusate concentration of LC was increased, CLHin

showed a significant reduction (p < 0.01) when the concentration of LC was 200 and 500 µM

(Table 2). In addition, the Kin parameter also showed a significant reduction (p< 0.01) at LC

concentration of 200 and 500 µM (Table 2).

Reproducibility and reversed concentration-ranging studies

The viability and reproducibility of the perfused liver preparation was assessed in two

livers by performing 4 Periods 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 4 Periods (EH, 0.130 ± 0.013; Kin 0.042 ± 0.005 sec-1; CLHin 0.097 ± 0.013 ml • sec-

1) 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 2-4 with a decreasing concentrations of unlabeled PLC (from 50 µM to 1.0 µM) were

carried out. With no background PLC in the perfusate, the EH (0.099), Kin (0.043 sec-1) and

CLHin (0.102 ml • sec-1) 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 (EH, 0.072; Kin 0.028 sec-1; CLHin 0.063 ml • sec-1) to values which were close to

those for the PLC concentration-ranging study where the concentration of unlabeled PLC in

perfusate was 50 µM in Period 4 (Table 1).


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Discussion 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 were

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 EH 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 to the limited capacity of the

hepatic intracellular enzymes to mediate their metabolism. In the present study, data from

impulse-response 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 (Pessotto et

al. 1996, Longo 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 EH 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


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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 CLHin being

substantially less than perfusate flow rate (5.7 vs 30 ml • min-1; Table 1) suggests that uptake

of PLC into 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 following

parameters Kin, Kout and Kseq which correspond to the influx, efflux and sequestration rate

constants, respectively (Goresky et al. 1993). During IR experiments with PLC, the influx rate

constant Kin, was well defined by the data, whereas the estimate of Kout and Kseq were not

able to be estimated. With the Kout and Kseq 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


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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

behaviour was not observed for PLC which presents an upward straight line constant for the

first 16 sec (p < 0.001; r2 = 0.992; Fig. 3).

The low values of EH, 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 Kin, CLHin and EH 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 Kin, CLHin and EH across

Periods 1 to 4 may be due to decreasing viability of the liver preparation over the

experimental period, or an accumulating effect of increasing background concentration of

PLC through the Periods 2-4. 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 Kin (0.028 sec-1), CLHin (0.063 ml • sec-1), and EH (0.072)

of PLC are comparable to 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, γ-

butyrobetaine, share the same uptake transport system (Christiansen and Bremer 1976;

Nakajima et al. 1999; Yokogawa et al. 1999). Since it is likely that LC also shares the same

transport system with short-chain carnitine esters in other isolated perfused organs


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(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 Kin and CLHin values for PLC

decreased significantly (p < 0.05). This observation demonstrates 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 LC transport in cell membranes is performed by a family of

organic transporters designated organic/carnitine transporter (Octn) (Tamai et al. 1997;

Koepsell, 2004). Some novel members of this family, namely Octn1, -2, -3 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,

resulted also able to transport the short-chain acyl esters of LC such as acetyl- and propionyl-

L-carnitine in cells expressing this transporter (Wu et al. 1999). Interestingly, the authors have

shown that the Octn2-mediated transport for acetyl- and propionyl-L-carnitine was inhibited

by LC 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. 2001). In contrast, a low level (but detectable)

expression of Octn2 has been reported in the liver (Tamai et al. 1998; Slitt et al. 2002;


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Choudhuri et al. 2003). The low expression of Octn2 in liver could explain the low EH of both

LC and PLC observed during the perfusion of these compounds in both the present and

previous study (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 demonstrated that for LC the entry into

hepatocytes is very low. For PLC the EH, Kin and CLHin 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.


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Footnotes: This work was financially supported by Sigma Tau SpA, Pomezia, Rome,

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Legends for figures.

Figure 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

Figure 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.

Figure 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 represents the mean ± SD of 4 different experiments.

Figure 4. Relationship between extraction ratio and influx rate constant (sec-1) of [14C]PLC in

concentration-ranging study with PLC (p < 0.001; r2 = 0.637).


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TABLE 1Effect of increasing concentration of PLC in perfusate on the hepatic extraction and influx parameter

for radiolabeled PLCa

Perfusate PLC concentration (µM)Parameter

0.0 1.0 5.0 50.0

Hepatic extraction

ratio

0.141 ± 0.029 0.126 ± 0.025 0.104 ± 0.019* 0.067 ± 0.017**

Influx rate constant

(sec-1)

0.037 ± 0.009 0.031 ± 0.008 0.030 ± 0.008 0.021 ± 0.004**

Hepatic influx

clearance (ml • sec-1)

0.095 ± 0.016 0.080 ± 0.012 0.076 ± 0.013* 0.051 ± 0.006**

aData are mean ± S.D. of four experiments. The definition of the parameters is shown in Materials

and Methods. * p<0.05; ** p<0.01, significantly different from the 0 µM concentration.

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

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TABLE 2Effect of increasing concentration of LC in perfusate on the hepatic extraction and influx parameter for

radiolabeled PLCa

Perfusate LC concentration (µM)Parameter

0.0 50.0 200.0 500.0

Hepatic extraction

ratio

0.092 ± 0.050 0.052 ± 0.065 0.029 ± 0.074 0.029 ± 0.060

Influx rate constant

(sec-1)

0.045 ± 0.009 0.041 ± 0.015 0.025 ± 0.012** 0.022 ± 0.009**

Hepatic influx

clearance (ml • sec-1)

0.104 ± 0.018 0.092 ± 0.024 0.059 ± 0.019** 0.052 ± 0.014**

aData are mean ± S.D. of four experiments. The definition of the parameters is shown in Materials andMethods. ** p<0.01 significantly different from the 0 µM concentration.




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