Direct evidence of the temperature dependence of Gd-BOPTA transport in the intact rat liver

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Article

Reference

Direct evidence of the temperature dependence of Gd-BOPTA transport in the intact rat liver

PLANCHAMP MESSEILLER, Corinne, et al.

Abstract

The aim was to study the influence of temperature on the transport of the hepatobiliary contrast agent Gadobenate dimeglumine (Gd-BOPTA). Rat livers were isolated and perfused with Gd-BOPTA at 12, 25, 30, 36 and 38 degrees C. After the perfusion period, biopsies were collected and the MR signal intensity was measured. Uptake and biliary excretion were quantified with radiolabeled Gd-BOPTA. MR signal intensity decreased with temperature of perfusion. This phenomenon was appropriately quantified with 153Gd and 153Sm labeling, in contrast to 67Ga.

PLANCHAMP MESSEILLER, Corinne, et al . Direct evidence of the temperature dependence of Gd-BOPTA transport in the intact rat liver. Applied Radiation and Isotopes , 2005, vol. 62, no.

6, p. 943-9

DOI : 10.1016/j.apradiso.2004.11.002 PMID : 15799874

Available at:

http://archive-ouverte.unige.ch/unige:40294

Disclaimer: layout of this document may differ from the published version.

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Applied Radiation and Isotopes 62 (2005) 943–949

Direct evidence of the temperature dependence of Gd-BOPTA transport in the intact rat liver

Corinne Planchamp , Gerd J. Beyer, Daniel O. Slosman, Franc - ois Terrier, Catherine M. Pastor

Laboratoire de physiopathologie he´patique, et imagerie mole´culaire, De´partement de Radiologie, Hoˆpitaux Universitaires de Gene`ve, Rue Micheli-du-Crest 24, CH-1211 Gene`ve 14, Switzerland

Received 18 June 2004; received in revised form 12 November 2004; accepted 18 November 2004

Abstract

The aim was to study the influence of temperature on the transport of the hepatobiliary contrast agent Gadobenate dimeglumine (Gd-BOPTA). Rat livers were isolated and perfused with Gd-BOPTA at 12, 25, 30, 36 and 381C. After the perfusion period, biopsies were collected and the MR signal intensity was measured. Uptake and biliary excretion were quantified with radiolabeled Gd-BOPTA. MR signal intensity decreased with temperature of perfusion. This phenomenon was appropriately quantified with¹⁵³Gd and¹⁵³Sm labeling, in contrast to⁶⁷Ga.

Keywords:Cellular imaging; Gd-BOPTA; Hepatocytes; Isolated perfused rat liver; Magnetic resonance imaging; Radiolabeling

1. Introduction

Gadobenate dimeglumine (Gd-BOPTA) is a new hepatobiliary contrast agent for magnetic resonance imaging (MRI). Its structure differs from the long used extracellular contrast agent gadopentetate dimeglumine (Gd-DTPA) by an additional lipophilic moiety (Fig. 1).

With this lipophilic substituent, Gd-BOPTA is taken up speciﬁcally by hepatocytes and is excreted into the bile.

Consequently, Gd-BOPTA has a dual imaging capabil- ity. On the one hand, it is used as an extracellular contrast agent in the immediate postinjection phase of contrast enhancement to reveal hypervascular regions, and on the other hand, it enters into hepatocytes and

facilitates MRI detection and characterization of hepatic diseases in a later delayed phase (Hamm et al., 1999;

Kuwatsuru et al., 2001;Manfredi et al., 1998;Schneider et al., 2003).

After diffusion into the extracellular space, part of Gd-BOPTA is taken up into hepatocytes and, after trafﬁcking across the cell, is excreted into the bile without biotransformation (Lorusso et al., 1999;Schuh- mann-Giampieri et al., 1993). In rats, 50% of the dose injected is excreted into the bile, the remaining being excreted into urine (De Hae¨n et al., 1996;Lorusso et al., 1999). In contrast, only 2–7% of the dose is excreted into the bile in humans (Lorusso et al., 1999;Spinazzi et al., 1998). The transport of Gd-BOPTA into hepatocytes is not fully understood. Evidence exists that Gd-BOPTA enters rat hepatocytes through a transporter belonging to the organic anion transporting polypeptide family (Oatps) localized on the sinusoidal membrane of hepatocytes (Cle´ment et al., 1998; Hahn and Saini, www.elsevier.com/locate/apradiso

doi:10.1016/j.apradiso.2004.11.002

Corresponding author. Tel.: +41 22 372 93 53;

fax: +41 22 372 93 66.

E-mail address:[email protected] (C. Planchamp).

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1998; Pastor et al., 2003). After its intracellular transport, Gd-BOPTA is eliminated into the bile through the ATP-dependent multidrug resistance-associated protein 2 (Mrp2) at the canalicular membrane of the hepatocyte (De Hae¨n et al., 1996; Pascolo et al., 2001).

In the isolated perfused rat liver, it was shown that the increase in MR signal intensity during Gd-BOPTA perfusion can be recorded over time with a direct visualization of the absence of Gd-BOPTA entry in hepatocytes when livers were co-perfused with Gd- BOPTA and bromosulfophthalein (pharmacological inhibition) (Pastor et al., 2003). In a hollow-ﬁber bioreactor containing freshly isolated rat hepatocytes, we showed that the transport of Gd-BOPTA into hepatocytes can be successfully described by compart- mental analysis of the MR signal intensity recorded over time and supports the hypothesis of a transporter- mediated uptake (Planchamp et al., 2004a, b).

However, information is lacking on the long- and short-term regulation of Gd-BOPTA transport in hepatocytes. Although a temperature dependence of transport through Oatps and Mrps has been previously reported (Payen et al., 2000; Ruiz-Garcia et al., 2002;

Zamek-Gliszczynski et al., 2003), its inﬂuence on the MR signal is unknown. Consequently, the aim of our study was to determine, in the isolated perfused rat liver model, the inﬂuence of temperature on Gd-BOPTA transport and its consequences on the MR signal.

Because the MR signal does not allow exact quantiﬁca- tion of Gd-BOPTA concentration in tissues, the transport of radiolabeled Gd-BOPTA was also measured.

2. Materials and methods

2.1. Chemicals

Gd-BOPTA was provided by Bracco Research (Geneva, Switzerland). Gd-DTPA is commercially available (Magnevist, Schering, Germany). ¹⁵³GdCl₃ (1.0 GBq/mL) was obtained from Gamma-Service Iso- topen und Strahlentechnik GmbH (Leipzig, Germany).

67GaCl3(1.11 GBq/mL) was provided by Mallinckrodt (Zu¨rich, Switzerland).¹⁵³Sm was extracted from¹⁵³Sm- EDTMP (1.3 GBq/mL, Quadramet, Schering, Ger- many) as described below. All other chemicals were of analytical grade.

2.2. Preparation of radiochemicals

153Gd, ¹⁵³Sm, and ⁶⁷Ga isotopes were used to radiolabel Gd-DTPA and Gd-BOPTA. Contrast agents labeled with¹⁵³Gd were obtained by adding 0.5–4 MBq

153GdCl₃to 0.5 M solution (0.4 mL, 200mmol, pH 6.5–8) containing a slight excess of free ligand DTPA and BOPTA (0.01%). Labeling with ⁶⁷Ga was performed similarly using ⁶⁷GaCl₃. Contrast agents labeled with

153Sm were prepared from¹⁵³Sm-EDTMP after destruc- tion of the complex and re-complexation of the ¹⁵³Sm with the free BOPTA or DTPA in acid condition (¹⁵³Sm shows higher stability constants for BOPTA and DTPA chelate complexes as compared to those with EDTMP and only traces of¹⁵³Sm-EDTMP have been used). HCl (0.1 M, 0.1 mL) was added to ¹⁵³Sm-EDTMP (4 MBq) for decomplexation. Gd-DTPA and Gd-BOPTA 0.5 M solution (0.05 mL, 0.025 mmol) was added and the pH was checked. 0.1 M HCl (0.1 mL) was added before the addition of Gd-DTPA and Gd-BOPTA 0.5 M solution (0.35 mL, 0.175 mmol) if the pH exceeded 2.

Gd-DTPA and Gd-BOPTA (200mmol) labeled with any radioisotope were mixed with Krebs-Henseleit- bicarbonate (KHB) solution containing 118 mM NaCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 4.7 mM KCl, 26 mM NaHCO3, and 2.5 mM CaCl2 (1 L, 200mM). The presence of free ligand or free metal in the perfusion solution and in the outﬂow perfusate was determined by complexometry at pH 5.8, with xylenol orange as indicator (Schmitz et al., 1996). Brieﬂy, the radiolabeled solutions of contrast agent (0.4 mL) were mixed with a pH 5.8 buffer (10 mL) and xylenol orange indicator (0.4 mL, 0.03% w/V). A yellow color indicates the presence of a ligand excess while a violet color indicates a metal excess.

2.3. Animals

Before liver perfusion, Sprague-Dawley rats (300–450 g) were anesthetized with pentobarbital COO

COO

O

N

N COO

Gd ³⁺

- - -

- -

^{2 MGH}⁺

COO COO

COO

N

N COO

Gd ³⁺

-

- - -

2 MGH⁺

COO COO

COO

N

N COO

Gd

-

- - -

2 MGH

Gd-DTPA

Gd-BOPTA

Fig. 1. Chemical structure of Gd-DTPA and Gd-BOPTA.

MGH⁺stands for meglumine.

C. Planchamp et al. / Applied Radiation and Isotopes 62 (2005) 943–949 944

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(50 mg/kg, i.p.). The protocol was approved by the animal welfare committee of the University of Geneva and the veterinary ofﬁce and followed the guidelines for the care and use of laboratory animals.

2.4. Liver perfusion 2.4.1. Perfusion system

The entire perfusion system consisted of solution reservoir, pump (Ismatec, Glattbrugg-Zu¨rick, Switzer- land), heating circulator (ThermoHaake DC10/P5, Switzerland), oxygenator, ﬁlter, bubble trap, and temperature probe. The livers were perfused with KHB buffer during the entire protocol with a non-recirculat- ing perfusion. The perfusate was equilibrated in the oxygenator with a mixture of 95% O₂—% CO₂(2.5 L/

min) during the protocol. The temperature was measured continuously with a thermocouple thermometer (Extech Instruments Co, no 422315, Waltham, USA), the temperature probe being placed just upstream from the portal vein catheter.

2.4.2. Isolated perfused rat liver

Livers were perfused in situ as previously described (Pastor et al., 1996). Brieﬂy, the abdominal cavity was opened and the portal vein was cannulated with a G16 catheter (outer diameter: 1.8 mm) introduced into the portal vein up to 2–3 mm from the liver. A ligature was placed around the inferior vena cava above the left renal vein. After the cannulation of the portal vein, the abdominal vena cava was transected and KHB solution was pumped without delay into the portal vein. The ﬂow rate was slowly increased over 1 min up to 30 mL/

min. In a second step, the chest was opened and a second cannula (G14) inserted through the right atrium into the thoracic inferior vena cava and secured with a ligature. Finally, the ligature around the abdominal inferior vena cava was tightened. The KHB solution was perfused to the liver through the portal catheter and eliminated by the catheter placed in the thoracic inferior vena cava. For bile collection, the common bile duct was cannulated with a PE 10 catheter. Livers were perfused with KHB solution for 15 min (recovery period) and then with KHB solution+200mM radiolabeled contrast agent for 30 min. Outﬂow perfusate was collected every minute during 10 min and every 5 min during 20 min. After the 30-min perfusion period, 5 biopsies and the total bile volume were collected and the contrast agent content was determined by radioactivity measurements. The remaining liver tissue was weighed and used for MRI. During the perfusion, the liver viability was assessed by monitoring portal vein pressure (Hewlett Packard 78353B, Palo Alto, USA).

2.5. Magnetic resonance imaging

T1-weighted imaging of tubes containing at least 4 g of liver tissue was performed at room temperature on an Intera 1.5 T MR system (Philips Medical Systems, Cleveland, USA) using a fast ﬁeld echo sequence with the following imaging parameters: saturation prepulse, TR/TE 4.3/1.3 ms, FA 801, FOV 20 cm, matrix size 96128, slice thickness 20 mm. A coil used for human head imaging was used. Mean signal intensity was measured in a circular region of interest drawn inside the tubes.

2.6. Radioactivity measurements

Radioactivity (in counts per minute) was measured in the perfusion solution (KHB+contrast agents, 1 g), the outﬂow perfusate (1 g), in KHB solution free of contrast agent (blank, 1 g), the bubble trap (before entry in the liver, 1 g), the bile (50mg) and the liver biopsies (0.5–1 g) by a Packard Cobra Auto-Gamma counter (Canberra Packard, Switzerland). For concentration determina- tions, we assumed that density was 1 g/mL for all samples.

2.7. Experimental protocol

2.7.1. Radioisotopes and hepatic detection of contrast agents

Livers were perfused at 381C with radiolabeled Gd- DTPA that remains extracellular or with radiolabeled Gd-BOPTA that also enters into hepatocytes. Contrast agents were labeled with¹⁵³Gd,¹⁵³Sm or⁶⁷G a (n¼3 for each radioisotope).

2.7.2. Temperature and Gd-BOPTA transport

Livers were perfused with [¹⁵³Gd]Gd-BOPTA at 12, 25, 30, 36 and 381C (n¼3 for each temperature).

Additionally, a single liver in each group was perfused with [¹⁵³Gd]Gd-DTPA.

To calculate the apparent activation energy (E_a) required for the transport of contrast agent into the bile we used a modiﬁed Arrhenius equation

logB¼ E_a 2:30R

1

TþA; (1)

where Bis the amount of contrast agent excreted into the bile during 30 min,Eais the activation energy (kcal/

mol),Ris the gas constant (0.001987 kcal/mol K),T(K) is the absolute temperature (1C+273), and A is the Arrhenius constant. E_ais calculated from the slope of the linear regression logB¼fð1=TÞaccording to E_a¼ slope2:30R: (2)

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2.8. Statistical analysis

Data were expressed as mean7SD. Statistical analysis was performed with a Mann–WhitneyUtest or with a Kruskal–Wallis test when appropriate. A P value less than 0.05 was considered statistically signiﬁcant.

3. Results

3.1. Radioisotopes and hepatic detection of contrast agents

When livers were perfused with [¹⁵³Gd]Gd-DTPA (200mM, 381C), the concentration of Gd-DTPA in the liver was 23.372.9mM (equal to 0.1670.02% of the perfused dose) (Fig. 2A). The liver uptake was much

higher when livers were perfused with 200mM [¹⁵³Gd]Gd-BOPTA (441.6769.2mM, i.e. 2.970.5% of the perfused dose). For both contrast agents the concentration in the outﬂow perfusate rapidly increased and reached a similar concentration to the perfusion solution (Fig. 2B). However, the steady-state was signiﬁcantly lower during [¹⁵³Gd]Gd-BOPTA perfusion (18875mM) than during [¹⁵³Gd]Gd-DTPA perfusion (20071mM) (P¼0:046).

When contrast agents were labeled with¹⁵³Sm, similar results were obtained (Fig. 3A). 2.870.1% of the radioactivity perfused was measured in the liver after the 30-min perfusion with [¹⁵³Sm]Gd-BOPTA and only 0.1570.01% with [¹⁵³Sm]Gd-DTPA. In contrast, when contrast agents were labeled with⁶⁷Ga, the radioactivity in the liver was similar for Gd-DTPA and Gd-BOPTA, (0.2570.11%). The value found with [⁶⁷Ga]Gd-DTPA was similar to those obtained with Gd-DTPA labeled with¹⁵³Gd and¹⁵³Sm (P¼0:733).

MRI of the liver biopsies conﬁrmed that Gd-BOPTA entered into hepatocytes. The MR signal intensity was two times higher in tubes containing liver biopsies perfused with Gd-BOPTA than in those perfused with Gd-DTPA, independently of the radioisotope (Fig. 3B).

0 50 100 150 200 250

0 5 10 15 20 25 30

Gd-BOPTA Gd-DTPA 0

100 200 300 400 500 600

Gd-DTPA Gd-BOPTA

Concentration [µM]Concentration [µM]

time [min]

(A)

(B)

Fig. 2. (A) Contrast agent concentration measured by radioactivity in liver tissue after a 30-min perfusion of [¹⁵³Gd]Gd- DTPA and [¹⁵³Gd]Gd-BOPTA (200mM, 381C, 30 mL/min). (B) Time course of contrast agent concentrations in the outﬂow perfusate (mean7SD,n¼3).

SI ± SD: 43 ± 4 100 44 ± 8 100 50 ± 8 100 Radioactivityaccumulation [%ofperfuseddose/12gliverx30min]

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Gd-153 labeling Sm-153 labeling Ga-67 labeling (A)

(B)

Fig. 3. Inﬂuence of the radioisotope on the liver accumulation of DTPA and BOPTA complexes. (A) Radioactivity accumulation in livers perfused 30 min with Gd-DTPA and Gd-BOPTA (200mM, 381C, 30 mL/min) labeled with ⁶⁷Ga, ¹⁵³Sm, and

153Gd (mean7SD,n¼3). (B) MRI and MR signal intensity (SI) of liver tissue perfused 30 min with Gd-DTPA and Gd- BOPTA solution (200mM, 381C, 30 mL/min) labeled with

67Ga, ¹⁵³Sm, and ¹⁵³Gd. MRI of representative tubes are shown. As the liver tissue were imaged separately according to the radioisotope used, the signal intensities of Gd-BOPTA tubes were set to 100% and the signal intensities of Gd-DTPA tubes were calculated as a percentage of this value (n¼3).

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The complexometry analysis indicated that there was a slight excess of ligand (yellow color) in all solutions of perfusion containing radiolabeled contrast agent and outﬂow perfusate. This statement excludes the presence of free metal, which might result in unspeciﬁc binding in the liver.

3.2. Temperature and Gd-BOPTA transport

When biopsies from livers perfused at various temperatures were imaged, the signal intensity declined when the perfusion temperature diminished, allowing visualization of the decreasing transport of Gd-BOPTA into hepatocytes (Fig. 4). This ﬁnding was quantiﬁed by radioactivity measurements. At 381C, 2.970.5% of the amount of perfused [¹⁵³Gd]Gd-BOPTA accumulated in the liver and 4.071.0% in the bile (Fig. 5). In contrast, at 121C only 0.3070.01% was measured in the liver and no contrast agent was detected in the bile.

The logarithm of the amount of Gd-BOPTA measured in the bile was linearly correlated to the inverse function of the perfusion temperature (Fig. 5, r¼0:978; po0:005;

regression equation: y¼11468xþ38). According to the slope of the linear regression (1146871412), the apparent activation energy was 52.476.5 kcal/mol for Gd-BOPTA excretion into the bile.

4. Discussion

4.1. Radioisotopes and hepatic detection of contrast agents

As expected, the concentration of [¹⁵³Gd]Gd-BOPTA in the liver was more than two times higher than that in 12°C

20°C 30°C 36°C 38°C Temperature of liver perfusion

MR signal intensity at room temperature

40 ±5 60 ±7 69 ±3 91 ±1 100 MRI

Fig. 4. MRI of and MR signal intensity of tubes containing biopsies from livers perfused 30 min with Gd-BOPTA (200mM, 30 mL/min) at 12, 25, 30, 36 and 381C. MRI spectra of representative tubes are shown. The signal intensities in tubes containing tissue collected from livers perfused at 381C were set to 100% and the signal intensities of tubes containing tissue collected from livers perfused at lower temperature were calculated as percentage this value (mean7SD,n¼3).

0 1 2 3 4 5

0 5 10 15 20 25 30 35 40

temperature [°C]

Gd-BOPTA accumulation [% perfuseddose/ 12 g liver / 30 min]

liver bile

LogGd-BOPTAinthebile [%/12gliverx30min]

1000/temperature [1/ ˚K]

-3 -2 -1 0 1

3.2 3.3 3.4 3.5 3.6

Fig. 5. Amount of Gd-BOPTA measured in the liver and in the bile after a 30-min perfusion (200mM,¹⁵³Gd-labeling, 30 mL/min) as a function of the perfusion temperature (12, 25, 30, 36 and 381C, mean7SD,n¼3). The logarithm transformation was used to calculate the apparent activation energy (Ea) required for the transport of Gd-BOPTA into the bile according to the Arrhenius equation.

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the perfusion solution, indicating hepatocyte uptake of Gd-BOPTA (Fig. 2). In contrast, [¹⁵³Gd]Gd-DTPA concentration was much lower, because Gd-DTPA distributes only in the extracellular volume and not in hepatocytes. The lower concentration of [¹⁵³Gd]Gd- BOPTA measured in the outﬂow perfusate (188mM) compared to that of [¹⁵³Gd]Gd-DTPA (200mM) con- ﬁrms the hepatocyte uptake of Gd-BOPTA. Such results have already been observed in a similar experimental model when 500mM Gd-BOPTA and Gd-DTPA were perfused and measured by inductively coupled plasma atomic emission spectrometry (Pastor et al., 2003).

Thus, ¹⁵³Gd substitutes for non-radioactive Gd and consequently, [¹⁵³Gd]Gd-BOPTA quantification accu- rately reflects the transport of Gd-BOPTA into hepatocytes and the bile excretion. Although Gd-BOPTA entry in hepatocytes can be observed by MRI, the exact hepatic concentrations of contrast agents can not be quantified by signal intensity. Indeed, contrast agents acts indirectly on the MR signal by shortening the relaxation times of surrounding protons and the same concentration of contrast agents can modify the signal differently according to the binding to surrounding proteins and local microviscosity (Cavagna et al., 1997). Consequently, both techniques are useful to better understand the cellular behavior of contrast agents.

When livers were perfused with [¹⁵³Sm]Gd-DTPA and [¹⁵³Sm]Gd-BOPTA, the radioactivity in the biopsies was identical to that found with [¹⁵³Gd]Gd-DTPA and [¹⁵³Gd]Gd-BOPTA (Fig. 3). [¹⁵³Sm]Gd-BOPTA accumulated in hepatocytes while [¹⁵³Sm]Gd-DTPA did not.

Because¹⁵³Sm-DTPA and¹⁵³Sm-BOPTA have the same cellular behavior as ¹⁵³Gd-DTPA and ¹⁵³Gd-BOPTA,

153Sm and probably all other radiolanthanides (elements with atomic numbers 57–71 which closely resemble one another chemically) can be used to radiolabel Gd- BOPTA. Interestingly, study of [¹⁴⁷Pm]Gd-BOPTA transport in plasma membrane vesicles has been successfully reported (Pascolo et al., 1999, 2001). Of note, ¹⁵³Sm also emits b radiation whose thera- peutic effects are used in cancer therapy (Seraﬁni, 2003) and the complexation of ¹⁵³Sm with BOPTA described in our model might be a way to direct¹⁵³Sm into hepatocytes.

As seen in Fig. 3, the ⁶⁷Ga radioactivity in liver biopsies after perfusion with [⁶⁷Ga]Gd-BOPTA and [⁶⁷Ga]Gd-DTPA were identical, and similar to those found in liver perfused with Gd-DTPA labeled with

153Sm and¹⁵³Gd, indicating that⁶⁷Ga-BOPTA did not enter into hepatocytes. In contrast, the MR signal intensity was two times higher in biopsies perfused with [⁶⁷Ga]Gd-BOPTA than in those perfused with [⁶⁷Ga]Gd-DTPA showing that Gd-BOPTA did enter into hepatocytes. Thus, the use of⁶⁷Ga for labeling is not appropriate to detect the intracellular uptake of Gd-

BOPTA. A different behavior of ⁶⁷Ga-complexes compared to lanthanide-complexes has already been demonstrated in a comparative kinetic study of simultaneously injected¹⁶⁷Tm- and⁶⁷Ga-citrate in normal mice and in mice with tumors (Beyer et al., 1978).

4.2. Temperature and Gd-BOPTA transport

Temperature variation modiﬁed Gd-BOPTA transport into hepatocytes and consequently the MR signal (Fig. 4). At low temperature (121C), Gd-BOPTA did not enter into hepatocytes and behaved as the extracellular contrast agent Gd-DTPA. Interestingly, even a small variation of temperature from 36 to 381C induced changes in Gd-BOPTA transport as evidenced by the signal intensity of the Gd-BOPTA enhanced images.

This observation may have clinical consequences as these temperatures correspond to values frequently measured in humans. Actually, a lower body temperature could result in a reduced uptake of Gd-BOPTA by normal hepatocytes and diminish the contrast with nonfunctioning hepatocytes that do not take up Gd-BOPTA (Kuwatsuru et al., 2001;Schneider et al., 2003).

The apparent activation energy obtained using the Arrhenius equation was 52.6 kcal/mol for excretion of Gd-BOPTA into bile. Our results conﬁrm the active excretion of Gd-BOPTA into bile. Indeed, high activation energies (420 kcal/mol) are commonly associated with active transport, while low activation energies are associated with passive diffusion or facilitated transport via an energy-independent carrier (Ruiz-Garcia et al., 2002). This ﬁnding is in good agreement with the ATP- dependence of the Mrp2-transporter (De Hae¨n et al., 1996;Pascolo et al., 2001).

In contrast to the amount of Gd-BOPTA measured in the bile that depends only on biliary excretion, the amount of Gd-BOPTA in the liver depends on both liver uptake and biliary excretion. Hence, the apparent activation energy of the uptake transport could not be determined with our data.

In summary, the isolated perfused rat liver showed that radiolabeling of contrast agents is accurate and useful to quantify contrast agents transport. Labeling with radiolanthanides such as ¹⁵³Gd and ¹⁵³Sm is appropriate because¹⁵³Gd-BOPTA and¹⁵³Sm-BOPTA are equivalent to Gd-BOPTA, in contrast to ⁶⁷Ga- BOPTA. Uptake of Gd-BOPTA into hepatocytes and biliary excretion are highly temperature dependent with important consequences on the MR signal intensity. The lower the temperature, the lower the transport, and the lower the MR signal intensity. Thus, the regulation of Gd-BOPTA transporters is important to consider for the interpretation of the signal intensity obtained during liver imaging.

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Acknowledgements

The authors thank Sophie Namy for excellent technical support, Marko K Ivancevic for the MRI and Sibylle Pochon & Herve´ Tournier (Bracco Re- search) for the Gd-BOPTA supply and information.

This work was supported by the ‘‘Fonds National Suisse de la Recherche Scientiﬁque’’ no 3200-100 868 to CM Pastor.

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