Application of Pharmacokinetic Modeling to Characterize Hepatobiliary Disposition of Imaging Agents and Alterations due to Liver Injury in Isolated Perfused Rat Livers.

BACKGROUND: Understanding the impact of altered hepatic uptake and/or efflux on the hepatobiliary disposition of the imaging agents [99mTc]Mebrofenin (MEB) and [153Gd]Gadobenate dimeglumine (BOPTA) is important for proper estimation of liver function. METHODS: A multi-compartmental pharmacokinetic (PK) model describing MEB and BOPTA disposition in isolated perfused rat livers (IPRLs) was developed. The PK model was simultaneously fit to MEB and BOPTA concentration-time data in the extracellular space, hepatocytes, bile canaliculi, and sinusoidal efflux in livers from healthy rats, and to BOPTA concentration-time data in rats pretreated with monocrotaline (MCT). RESULTS: The model adequately described MEB and BOPTA disposition in each compartment. The hepatocyte uptake clearance was much higher for MEB (55.3 mL/min) than BOPTA (6.67 mL/min), whereas the sinusoidal efflux clearance for MEB (0.000831 mL/min) was lower than BOPTA (0.0127 mL/min). The clearance from hepatocytes to bile (CLbc) for MEB (0.658 mL/min) was similar to BOPTA (0.642 mL/min) in healthy rat livers. The BOPTA CLbc was reduced in livers from MCT-pretreated rats (0.496 mL/min), while the sinusoidal efflux clearance was increased (0.0644 mL/min). CONCLUSION: A PK model developed to characterize MEB and BOPTA disposition in IPRLs was used to quantify changes in the hepatobiliary disposition of BOPTA caused by MCT pretreatment of rats to induce liver toxicity. This PK model could be applied to simulate changes in the hepatobiliary disposition of these imaging agents in rats in response to altered hepatocyte uptake or efflux associated with disease, toxicity, or drug-drug interactions.

Monocrotaline Toxicity Alters the Function of Hepatocyte Membrane Transporters in Rats.

Pyrrolizidine alkaloid monocrotaline (MCT) induces sinusoidal obstruction syndrome (SOS) in rats characterised by a sinusoidal congestive obstruction. Additionally, MCT administration decreases the biliary excretion of gadobenate dimeglumine (BOPTA), a hepatobiliary substrate used in clinical imaging. BOPTA crosses hepatocyte membranes through organic anion transporting polypeptides, multidrug-resistance-associated protein 2, and Mrp3/4 transporters, and a modified function of these transporters is likely to explain the decreased biliary excretion. This study compared BOPTA transport across hepatocytes in livers isolated from normal (Nl) rats and rats with intragastric administration of MCT. BOPTA hepatocyte influx clearance was similar in both groups, while biliary clearance and bile concentrations were much lower in MCT than in Nl livers. BOPTA efflux clearance back to the sinusoids compensated for the low biliary excretion, and hepatocyte concentrations remained similar in both groups. This SOS-associated changes of transporter functions might impact the pharmacokinetics of numerous drugs that use similar transporters to cross hepatocytes.

New Pharmacokinetic Parameters of Imaging Substrates Quantified from Rat Liver Compartments.

Hepatobiliary imaging is increasingly used by pharmacologists to quantify liver concentrations of transporter-dependent drugs. However, liver imaging does not quantify concentrations in extracellular space, hepatocytes, and bile canaliculi. Our study compared the compartmental distribution of two hepatobiliary substrates gadobenate dimeglumine [BOPTA; 0.08 liver extraction ratio (ER)] and mebrofenin (MEB; 0.93 ER) in a model of perfused rat liver. A gamma counter placed over livers measured liver concentrations. Livers were preperfused with gadopentetate dimeglumine to measure extracellular concentrations. Concentrations coming from bile canaliculi and hepatocytes were calculated. Transporter activities were assessed by concentration ratios between compartments and pharmacokinetic parameters that describe the accumulation and decay profiles of hepatocyte concentrations. The high liver concentrations of MEB relied mainly on hepatocyte and bile canaliculi concentrations. In contrast, the three compartments contributed to the low liver concentrations obtained during BOPTA perfusion. Nonlinear regression analysis of substrate accumulation in hepatocytes revealed that cellular efflux is measurable ∼4 minutes after the start of perfusion. The hepatocyte-to-extracellular concentration ratio measured at this time point was much higher during MEB perfusion. BOPTA transport by multidrug resistance associated protein 2 induced an aquaporin-mediated water transport, whereas MEB transport did not. BOPTA clearance from hepatocytes to bile canaliculi was higher than MEB clearance. MEB did not efflux back to sinusoids, whereas BOPTA basolateral efflux contributed to the decrease in hepatocyte concentrations. In conclusion, our ex vivo model quantifies substrate compartmental distribution and transport across hepatocyte membranes and provides an additional understanding of substrate distribution in the liver. SIGNIFICANCE STATEMENT: When transporter-dependent drugs target hepatocytes, cellular concentrations are important to investigate. Low concentrations on cellular targets impair drug therapeutic effects, whereas excessive hepatocyte concentrations may induce cellular toxicity. With a gamma counter placed over rat perfused livers, we measured substrate concentrations in the extracellular space, hepatocytes, and bile canaliculi. Transport across hepatocyte membranes was calculated. The study provides an additional understanding of substrate distribution in the liver.

Steatosis Alters the Activity of Hepatocyte Membrane Transporters in Obese Rats.

Fat accumulation (steatosis) in ballooned hepatocytes alters the expression of membrane transporters in Zucker fatty (fa/fa) rats. The aim of the study was to quantify the functions of these transporters and their impact on hepatocyte concentrations using a clinical hepatobiliary contrast agent (Gadobenate dimeglumine, BOPTA) for liver imaging. In isolated and perfused rat livers, we quantified BOPTA accumulation and decay profiles in fa/+ (normal) and fa/fa hepatocytes by placing a gamma counter over livers. Profiles of BOPTA accumulation and decay in hepatocytes were analysed with nonlinear regressions to characterise BOPTA influx and efflux across hepatocyte transporters. At the end of the accumulation period, BOPTA hepatocyte concentrations and influx clearances were not significantly different in fa/+ and fa/fa livers. In contrast, bile clearance was significantly lower in fatty hepatocytes while efflux clearance back to sinusoids compensated the low efflux into canaliculi. The time when BOPTA cellular efflux impacts the accumulation profile of hepatocyte concentrations was slightly delayed (2 min) by steatosis, anticipating a delayed emptying of hepatocytes. The experimental model is useful for quantifying the functions of hepatocyte transporters in liver diseases.

Concentrations and pharmacokinetic parameters of MRI and SPECT hepatobiliary agents in rat liver compartments.

BACKGROUND: In hepatobiliary imaging, systems detect the total amount of agents originating from extracellular space, bile canaliculi, and hepatocytes. They add in situ concentration of each compartment corrected by its respective volume ratio to provide liver concentrations. In vivo contribution of each compartment to liver concentration is inaccessible. Our aim was to quantify the compartmental distribution of two hepatobiliary agents in an ex vivo model and determine how their liver extraction ratios and cholestasis (livers lacking canalicular transporters) might modify it. METHODS: We perfused labelled gadobenate dimeglumine (Bopta, 200 µM, 7% liver extraction ratio) and mebrofenin (Meb, 64 µM, 94% liver extraction ratio) in normal (n = 18) and cholestatic (n = 6) rat livers. We quantified liver concentrations with a gamma counter placed over livers. Concentrations in hepatocytes and bile canaliculi were calculated. Mann-Whitney and Kruskal-Wallis tests were used. RESULTS: Hepatocyte concentrations were 2,043 ± 333 µM (Meb) versus 360 ± 69 µM (Bopta, p < 0.001). Meb extracellular concentrations did not contribute to liver concentrations (1.3 ± 0.3%). The contribution of Bopta extracellular concentration was 12.4 ± 1.9% (p < 0.001 versus Meb). Contribution of canaliculi was similar for both agents (16%). Cholestatic livers had no Bopta in canaliculi but their hepatocyte concentrations increased in comparison to normal livers. CONCLUSION: Hepatocyte concentrations are correlated to liver extraction ratios of hepatobiliary agents. When Bopta is not present in canaliculi of cholestatic livers, hepatocyte concentrations increase in comparison to normal livers. This new understanding extends the interpretation of clinical liver images.

Hepatocyte Concentrations of Imaging Compounds Associated with Transporter Inhibition: Evidence in Perfused Rat Livers.

In the liver, several approaches are used to investigate and predict the complex issue of drug-induced transporter inhibition. These approaches include in vitro assays and pharmacokinetic models that predict how inhibitors modify the systemic and liver concentrations of the victim drugs. Imaging is another approach that shows how inhibitors might alter liver concentrations stronger than systemic concentrations. In perfused rat livers associated with a gamma counter that measures liver concentrations continuously, we previously showed how fluxes across transporters generate the hepatocyte concentrations of two clinical imaging compounds, one with a low extraction ratio [gadobenate dimeglumine (BOPTA)] and one with a high extraction ratio [mebrofenin (MEB)]. BOPTA and MEB are transported by rat organic anion transporting polypeptide and multiple resistance-associated protein 2, which are both inhibited by rifampicin. The aim of the study is to measure how rifampicin modifies the hepatocyte concentrations and membrane clearances of BOPTA and MEB and to determine whether these compounds might be used to investigate transporter-mediated drug-drug interactions in clinical studies. We show that rifampicin coperfusion greatly decreases BOPTA hepatocyte concentrations, but increases those of MEB. Rifampicin strongly decreases BOPTA hepatic clearance. In contrast, rifampicin decreases moderately MEB hepatic clearance and blocks the biliary intrinsic clearance, increasing MEB hepatocyte concentrations. In conclusion, low concentrations prevent the quantification of BOPTA biliary intrinsic clearance, while MEB is a promising imaging probe substrate to evidence transporter-mediated drug-drug interactions when inhibitors act on influx and efflux transporters.

Isolated Perfused Rat Livers to Quantify the Pharmacokinetics and Concentrations of Gd-BOPTA.

With recent advances in liver imaging, the estimation of liver concentrations is now possible following the injection of hepatobiliary contrast agents and radiotracers. However, how these images are generated remains partially unknown. Most experiments that would be helpful to increase this understanding cannot be performed in vivo. For these reasons, we investigated the liver distribution of the magnetic resonance (MR) contrast agent gadobenate dimeglumine (Gd-BOPTA, MultiHance®, Bracco Imaging) in isolated perfused rat livers (IPRLs). In IPRL, we developed a new set up that quantifies simultaneously the Gd-BOPTA compartment concentrations and the transfer rates between these compartments. Concentrations were measured either by MR signal intensity or by count rates when the contrast agent was labelled by [153Gd]. With this experimental model, we show how the Gd-BOPTA hepatocyte concentrations are modified by temperature and liver flow rates. We define new pharmacokinetic parameters to quantify the canalicular transport of Gd-BOPTA. Finally, we present how transfer rates generate Gd-BOPTA concentrations in rat liver compartments. These findings better explain how liver imaging with hepatobiliary radiotracers and contrast agents is generated and improve the image interpretation by clinicians.

Quantification of hepatic perfusion and hepatocyte function with dynamic gadoxetic acid-enhanced MRI in patients with chronic liver disease.

The purpose of the present study was to develop and perform initial validation of dynamic MRI enhanced with gadoxetic acid as hepatobiliary contrast agent to quantify hepatic perfusion and hepatocyte function in patients with chronic liver disease. Free-breathing, dynamic gadoxetic acid-enhanced MRI was performed at 3.0 T using a 3D time-resolved angiography sequence with stochastic trajectories during 38 min. A dual-input three-compartment model was developed to derive hepatic perfusion and hepatocyte function parameters. Method feasibility was assessed in 23 patients with biopsy-proven chronic liver disease. Parameter analysis could be performed in 21 patients (91%). The hepatocyte function parameters were more discriminant than the perfusion parameters to differentiate between patients with minimal fibrosis (METAVIR F0-F1), intermediate fibrosis (F2-F3) and cirrhosis (F4). The areas under the receiver operating characteristic curves (ROCs) to diagnose significant fibrosis (METAVIR F ≥ 2) were: 0.95 (95% CI: 0.87-1; P<0.001) for biliary efflux, 0.88 (95% CI: 0.73-1; P<0.01) for sinusoidal backflux, 0.81 (95% CI: 0.61-1; P<0.05) for hepatocyte uptake fraction and 0.75 (95% CI: 0.54-1; P<0.05) for hepatic perfusion index (HPI), respectively. These initial results in patients with chronic liver diseases show that simultaneous quantification of hepatic perfusion and hepatocyte function is feasible with free breathing dynamic gadoxetic acid-enhanced MRI. Hepatocyte function parameters may be relevant to assess liver fibrosis severity.

Gadoxetate-enhanced MR imaging and compartmental modelling to assess hepatocyte bidirectional transport function in rats with advanced liver fibrosis.

OBJECTIVES: Changes in the expression of hepatocyte membrane transporters in advanced fibrosis decrease the hepatic transport function of organic anions. The aim of our study was to assess if these changes can be evaluated with pharmacokinetic analysis of the hepatobiliary transport of the MR contrast agent gadoxetate. METHODS: Dynamic gadoxetate-enhanced MRI was performed in 17 rats with advanced fibrosis and 8 normal rats. After deconvolution, hepatocyte three-compartmental analysis was performed to calculate the hepatocyte influx, biliary efflux and sinusoidal backflux rates. The expression of Oatp1a1, Mrp2 and Mrp3 organic anion membrane transporters was assessed with reverse transcription polymerase chain reaction. RESULTS: In the rats with advanced fibrosis, the influx and efflux rates of gadoxetate decreased and the backflux rate increased significantly (p = 0.003, 0.041 and 0.010, respectively). Significant correlations were found between influx and Oatp1a1 expression (r = 0.78, p < 0.001), biliary efflux and Mrp2 (r = 0.50, p = 0.016) and sinusoidal backflux and Mrp3 (r = 0.61, p = 0.002). CONCLUSION: These results show that changes in the bidirectional organic anion hepatocyte transport function in rats with advanced liver fibrosis can be assessed with compartmental analysis of gadoxetate-enhanced MRI. KEY POINTS: • Expression of hepatocyte transporters is modified in rats with advanced liver fibrosis. • Kinetic parameters at gadoxetate-enhanced MRI are correlated with hepatocyte transporter expression. • Hepatocyte transport function can be assessed with compartmental analysis of gadoxetate-enhanced MRI. • Compartmental analysis of gadoxetate-enhanced MRI might provide biomarkers in advanced liver fibrosis.

Liver Perfusion Modifies Gd-DTPA and Gd-BOPTA Hepatocyte Concentrations Through Transfer Clearances Across Sinusoidal Membranes.

BACKGROUND AND OBJECTIVES: Gadobenate dimeglumine (Gd-BOPTA) is a commercialised hepatobiliary contrast agent used during liver magnetic resonance imaging (MRI) to detect liver diseases. It enters into human hepatocytes through organic anion transporting polypeptides (OATP1B1/B3) and crosses the canalicular transporter multiple resistance-associated protein 2 (MRP2) to be excreted into bile canaliculi. Gd-BOPTA can return to sinusoids via the sinusoidal transporters MRP3/MRP4. Hepatocyte concentrations of Gd-BOPTA depend on three clearances: the sinusoidal clearance or volume of sinusoidal blood cleared of drugs per unit of time and two hepatocyte clearances (into bile canaliculi or back to sinusoids) or volume of hepatocytes cleared of drugs per unit of time in the respective liver compartments. The present study investigates whether changing liver blood flow modifies hepatocyte concentrations when plasma concentrations do not change. METHODS: We perfused normal rat livers at various portal flow rates (24, 30, and 36 ml/min) with 200 µM Gd-BOPTA and measured sinusoidal clearances, hepatocyte clearances, and hepatocyte concentrations of Gd-BOPTA. RESULTS: We showed that varying portal flow rates changes the sinusoidal clearance of Gd-BOPTA despite its low extraction ratio. Portal flow rates do not modify Gd-BOPTA clearance from hepatocytes into bile canaliculi but can change hepatocyte clearance back to sinusoids. CONCLUSION: At a given perfused concentration, portal flow rates modify Gd-BOPTA hepatocyte concentrations, a result important to consider when interpreting liver imaging.

Hepatocyte Concentrations of Indocyanine Green Reflect Transfer Rates Across Membrane Transporters.

Perioperative imaging with indocyanine green (ICG) is developing to increase safety in dissecting anatomical structures during hepatobiliary surgery. Images obtained with the fluorescence camera rely on concentrations measured in liver regions of interest. However, how ICG sinusoidal uptake and hepatocyte elimination rates generate ICG hepatocyte concentrations is largely unknown. To investigate such issue and better understand the role of membrane transporters in generating ICG hepatocyte concentrations, we perfused ICG in livers isolated from normal livers. Whether the well-known transporter inhibitor rifampicin modifies hepatocyte ICG concentrations was also studied. The dye has a very high and constant extraction ratio (96%) into hepatocytes. This persistent high extraction ratio generates a huge uphill concentration gradient across the sinusoidal membrane: from 5 µM (sinusoids) to 1600 µM (liver). When inside hepatocytes, ICG has low hepatocyte elimination (7 nmol/min.) and liver concentrations do not decrease much over time. Moreover, the tiny hepatocyte ICG efflux is mainly due to ICG return back to sinusoids (90%). Rifampicin slightly inhibits ICG uptake into hepatocytes and when inside hepatocytes blocks ICG efflux into bile canaliculi. In contrast, it increases ICG efflux back to sinusoids with significant decrease in ICG liver concentrations. Imaging with ICG in the perioperative period reflects the high hepatocyte concentrations and relies on the high extraction ratio across hepatocyte sinusoidal membrane. Although ICG concentrations are low in bile ducts, they are adequate for a good visualization and avoid bile duct injury.

How transfer rates generate Gd-BOPTA concentrations in rat liver compartments: implications for clinical liver imaging with hepatobiliary contrast agents.

Following the injection of hepatobiliary contrast agents, MRI detects all molecules included in a region of interest but cannot estimate true concentrations in sinusoids, interstitium, hepatocytes or bile canaliculi. The aim of the study was to measure true concentrations in hepatocytes and to show how transfer rates across sinusoidal and canalicular membranes generate these concentrations. We perfused livers isolated from normal rats with 200 µM Gd-DTPA and Gd-BOPTA and measured clearances from sinusoids to liver and from hepatocytes to bile canaliculi or back to interstitium. We detected Gd-BOPTA with a gamma probe and determined true concentrations in each liver compartment knowing their liver volumes. No pharmacokinetic modelling was applied. Gd-BOPTA clearance from sinusoids to liver (2.5 ± 0.4 mL/min) was 50 times higher than that of Gd-DTPA (0.05 ± 0.02 mL/min) when portal flow rate was 30 mL/min (p < 0.0001). Gd-BOPTA clearance from sinusoids to liver was always superior to hepatocyte clearance, and hepatocyte Gd-BOPTA concentrations measured by the probe increased over time. Gd-BOPTA concentrations reached 439 ± 83 µM in hepatocytes and 15 × 700 ± 3100 µM in bile canaliculi, while concentrations in sinusoids were 200 µM. Gd-BOPTA true concentrations in hepatocytes depend on the simultaneous clearances from sinusoids to hepatocytes and from hepatocytes to bile canaliculi and back to sinusoids. The study better defines how signal intensities are generated when hepatobiliary contrast agents are injected in clinical imaging. Copyright © 2016 John Wiley & Sons, Ltd.

Insights into the diagnosis of hepatocellular carcinomas with hepatobiliary MRI.

The incidence of hepatocellular carcinomas (HCCs) has increased worldwide in line with an improved screening by high-resolution imaging of cirrhotic livers. Besides abdominal ultrasonography and computerised tomography, magnetic resonance imaging (MRI) is an important tool to detect HCCs. With commercialisation of MR hepatobiliary contrast agents that cross membrane transporters in hepatocytes or tumour cells, MRI adds new information to detect and characterise HCCs. When tumour cells lose organic anion transporting polypeptides (OATP1B1/B3) in cell membranes facing sinusoidal blood, tumours appear hypointense (decreased contrast agent concentrations) in comparison to surrounding normal or cirrhotic liver that retains OATP1B1/B3 expression. However, expression, regulation, and prognostic significance of transporter evolution along carcinogenesis are not completely known. Moreover, understanding signal intensities in focal lesions also relies on transport functions of cellular efflux transporters. This manuscript reviews all the publications that associate liver imaging with hepatobiliary contrast agents and expression of transporters. The regulation of transporters along carcinogenesis to anticipate the prognosis of focal lesions is also included.

Advanced fibrosis: Correlation between pharmacokinetic parameters at dynamic gadoxetate-enhanced MR imaging and hepatocyte organic anion transporter expression in rat liver.

PURPOSE: To compare the value of enhancement and pharmacokinetic parameters measured at dynamic gadoxetate-enhanced magnetic resonance (MR) imaging in determining hepatic organic anion transporter expression in control rats and rats with advanced liver fibrosis. MATERIALS AND METHODS: Institutional animal review board approval was received before the study began. Advanced liver fibrosis was created in rats by means of carbon tetrachloride injections over an 8-week period. In 17 rats with liver fibrosis and eight control rats, dynamic gadoxetate-enhanced MR images of the liver were obtained during 1 hour after injection of 0.025 mmol gadoxetate per kilogram of body weight. Enhancement parameters (maximum enhancement [Emax], time to peak [Tmax], and elimination half-life) were measured on enhancement-versus-time curves, and pharmacokinetic parameters (hepatic extraction fraction [HEF] and mean residence time [MRT]) were obtained by means of deconvolution analysis of the concentration-versus-time curves in the liver and the portal vein. The parameters were correlated at simple and multiple regression analysis with the expression of the hepatic anion uptake transporter organic anion-transporting polypeptide 1A1 (Oatp1a1), the hepatobiliary transporter multidrug resistance-associated protein 2 (Mrp2), and the backflux transporter Mrp4, as determined with reverse transcription polymerase chain reaction. RESULTS: In rats with advanced liver fibrosis, the Emax, Tmax, HEF, and MRT decreased significantly relative to those in control rats, whereas the elimination half-life increased significantly. The enhancement and pharmacokinetic parameters correlated significantly with the expression of the transporters at simple regression analysis. At multiple regression analysis, HEF was the only parameter that was significantly associated with the expression of Oatp1a1 and Mrp2 (P < .001, r = 0.74 and P < .001, r = 0.70, respectively). CONCLUSION: The pharmacokinetic parameter HEF at dynamic gadoxetate-enhanced MR imaging is independently correlated with hepatic organic anion transporter expression.

Quantification of drug transport function across the multiple resistance-associated protein 2 (Mrp2) in rat livers.

To understand the transport function of drugs across the canalicular membrane of hepatocytes, it would be important to measure concentrations in hepatocytes and bile. However, these concentration gradients are rarely provided. The aim of the study is then to measure these concentrations and define parameters to quantify the canalicular transport of drugs through the multiple resistance associated-protein 2 (Mrp2) in entire rat livers. Besides drug bile excretion rates, we measured additional parameters to better define transport function across Mrp2: (1) Concentration gradients between hepatocyte and bile concentrations over time; and (2) a unique parameter (canalicular concentration ratio) that represents the slope of the non-linear regression curve between hepatocyte and bile concentrations. This information was obtained in isolated rat livers perfused with gadobenate dimeglumine (BOPTA) and mebrofenin (MEB), two hepatobiliary drugs used in clinical liver imaging. Interestingly, despite different transport characteristics including excretion rates into bile and hepatocyte clearance into bile, BOPTA and MEB have a similar canalicular concentration ratio. In contrast, the ratio was null when BOPTA was not excreted in bile in hepatocytes lacking Mrp2. The canalicular concentration ratio is more informative than bile excretion rates because it is independent of time, bile flows, and concentrations perfused in portal veins. It would be interesting to apply such information in human liver imaging where hepatobiliary compounds are increasingly investigated.

The role of organic anion transporters in diagnosing liver diseases by magnetic resonance imaging.

The expression and transport functions of organic anion transporters are modified in liver diseases, and therefore the vascular clearances of endogenous and exogenous organic anions that are taken up by these transporters have been used to assess liver diseases in patients. More recently, liver imaging with hepatobiliary contrast agents, tracers, and dyes that cross hepatocytes through the organic anion transporting polypeptides (OATPs)-multidrug resistance-associated proteins (MRPs) pathway were developed to detect and characterize focal lesions and to assess the severity of diffuse liver diseases. This review focuses mainly on magnetic resonance imaging and highlights the growing interest in imaging the OATPs-MRP2 pathway to better understand liver diseases. Imaging provides noninvasive measurements of tissue concentrations that result from the interplay between influx and efflux membrane transport systems in normal or injured hepatocytes. Imaging with magnetic resonance hepatobiliary contrast agents improves the detection and the characterization of hepatic focal lesions. New developments of imaging to assess liver function and understand the hepatocellular concentrations of contrast agents are discussed.