Article Figures & Data
Figures
- Fig. 1.
(A) Perfused rat liver. Livers were perfused with a nonrecirculating system. QH was 30 ml/min. Cin values were constant during substrate perfusion: 200 µM (BOPTA) and 64 µM (MEB). Cout values were collected. Cbile and Qbile were measured in the common bile duct. A gamma counter placed over a right liver lobe recorded Cliver. (B) Liver concentrations measured by the counter. Livers (n = 6) were perfused with KHB solution + 200 µM [153Gd]DTPA, KHB, KHB + 200 µM [153Gd]BOPTA, and KHB. During BOPTA perfusion, DTPA concentrations were reported (***). (C) BOPTA and MEB transport by Oatp, Mrp2, and Mrp3. Cout included substrates that did not enter into hepatocytes (black points) and those that entered and returned to sinusoids (brown asterisks). Substrates eliminated into bile canaliculi (red points).
- Fig. 2.
(A) BOPTA and MEB liver extraction ratios. (B) Qbile measured during BOPTA and MEB perfusion. (C) Compartmental distribution of BOPTA in normal livers (n = 6) perfused with KHB solution + 200 µM [153Gd]BOPTA (45–75 minutes) (D) Compartmental distribution of MEB in normal livers (n = 6) perfused with KHB + 64 µM [99mTc]MEB (45–75 minutes). Liver concentrations (black symbols) were measured by a gamma counter. Concentrations in extracellular compartment (red symbols) were measured during the previous DTPA perfusion. Concentrations that originate from the bile canaliculi (blue symbols) and from hepatocyte volume (green symbols) were calculated.
- Fig. 3.
BOPTA (A) and MEB (B) hepatocyte influx rate (vin, left y-axis, black circles) and bile excretion rate (vbile) plus basolateral efflux rate (vef) (right y-axis, open circles) during accumulation period (45–75 minutes). Normal livers (n = 12) were perfused with KHB solution + 200 µM [153Gd]BOPTA or KHB + 64 µM [99mTc]MEB. Difference between vin and vbile + vef (gray area). (C) Accumulation of BOPTA and MEB hepatocyte concentrations (from 45 to 75 minutes of the experimental protocol). Accumulation was best described by a hinge function (red curve) defined by two lines with a gentle connection between them that intersected at T0 defined as the time when cellular excretion affects hepatocyte concentrations. (D) Bile concentrations measured 5 minutes after the start of substrate perfusion (time 50 minutes of the experimental protocol).
- Fig. 4.
(A) BOPTA and MEB hepatocyte concentrations during the decay period (75–105 minutes) when livers were perfused with Krebs-Henseleit bicarbonate solution (rinse period). The decline in concentrations was best described by a two-phase decay (red curves). (B) BOPTA and MEB bile concentration decay.
- Fig. 5.
BOPTA (A) and MEB (B) CLbile (mlHC/min). CLbile was the slope of the linear regression between vbile and CHC during the accumulation and decay periods. BOPTA (C) and MEB (D) CLef (mlHC/min). CLef was the slope of linear regression between vef and CHC during the rinse period.
Tables
Imaging substrates P BOPTA MEB Concentrations (µM) End of perfusion period Cin 200 64 Cout 185 ± 3 5 ± 2 0.002 Cliver 574 ± 88 2,486 ± 362 0.002 CEC 62 ± 20 32 ± 7 0.004 CHC78% 441 ± 78 2,036 ± 389 0.002 CHC100% 566 ± 99 2,610 ± 498 0.002 CBC 72 ± 9 417 ± 49 0.002 Cbile 16,791 ± 2,085 97,017 ± 11,289 0.002 Transfer rates (nmol/min) End of perfusion period v 464 ± 80 1,797 ± 61 0.002 vin 561 ± 81 1,819 ± 54 0.002 vbile (nmol/min) 390 ± 84 990 ± 166 0.002 vef 97 ± 7 22 ± 8 0.002 Clearance parameters End of perfusion period CLH (mlKHB/min) 2.3 ± 0.4 27.8 ± 0.9 0.002 CLin (mlKHB/min) 2.8 ± 0.4 28.1 ± 0.8 0.002 Perfusion and rinse periods CLbile (mlHC/min) 0.89 ± 0.25 0.47 ± 0.15 0.009 Rinse period CLef (mlHC/min) 0.18 ± 0.03 0.01 ± 0.01 0.002 CLbile + CLef (mlHC/min) 1.1 ± 0.3 0.5 ± 0.1 0.004 Concentration ratios RHC/EC 4 ± 2 33 ± 19 0.002 Rbile/HC 31 ± 6 45 ± 12 0.03 RHV/HC 0.006 ± 0.001 0.0003 ± 0.0001 0.002 Rbile/EC 299 ± 111 3218 ± 1109 0.002 CHC100% is CHC78% multiplied by 100/78. Substrate removal rate from sinusoids (v), hepatocyte influx rate (vin), bile excretion rate (vbile), and efflux rate back into sinusoids (vef). Hepatocyte-to-extracellular concentration ratio (RHC/EC) were measured at T0 in the absence of hepatocyte efflux. Bile-to-hepatocyte concentration ratio (Rbile/HC) was the slope of the relationship between CHC (x-axis) and Cbile (y-axis) during the perfusion and rinse periods. Hepatic vein–to-hepatocyte concentration ratio (RHV/HC) was the slope of the relationship between CHC (x-axis) and Cef (y-axis) during the rinse period. Bile-to-extracellular concentration ratio (Rbile/EC) was the maximal ability of transporters to concentrate substrates from the EC to bile compartment at the end of the perfusion period. CLH and CLin were expressed in ml of Krebs-Henseleit Bicarbonate (KHB) solution/min (mlKHB/min). CLef and CLbile were expressed in ml of hepatocytes (HC)/min (mlHC/min).
Imaging substrate P BOPTA MEB Accumulation T0 (min) 4.3 ± 1.0 3.9 ± 1.4 0.54 L1 slope (µM/min) 70 ± 15 425 ± 82 0.004 L2 slope (µM/min) 10 ± 3 24 ± 6 0.004 Decay Y0 (µM) 588 ± 89 2731 ± 499 0.002 Y0,fast (µM) 327 ± 61 1723 ± 328 0.004 kfast,HC (min−1) 0.25 ± 0.19 0.21 ± 0.02 0.39 T1/2fast (min) 3.2 ± 1.6 3.3 ± 0.3 0.66 Y0,slow (µM) 235 ± 55 1008 ± 308 0.004 kslow,HC (min−1) 0.044 ± 0.007 0.016 ± 0.004 0.004 T1/2slow (min) 16 ± 3 48 ± 17 0.004 Rate constant ratio 6.4 ± 3.4 14.5 ± 4.5 0.02 Hepatocyte accumulation was best described by a hinge function that included a first line L1 for time below T0 and a second line L2 for time higher than T0. T0 was the time when the two lines would intersect if there were no curve connecting them. Before T0, L1 slope characterized BOPTA and MEB influx by Oatps. After T0, accumulation rates decreased due to concomitant entry and efflux from hepatocytes. During decay, hepatocyte concentrations were best described with a two-phase decay model. The model was the sum of two components working simultaneously and defined by rate constants kfast,HC and kslow,HC and starting Y values (Y0,fast and Y0,slow). For each liver, Y0 is Y0,fast + Y0,slow.
