Hepatic Clearance and Drug Metabolism Using Isolated Perfused Rat Liver

Yong Liu1, Steven J. Weber1, Emmanuel T. Onua2

1 Pfizer Global Research & Development, Ann Arbor, Michigan, 2 DeCODE Genetics, Woodridge, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.9
DOI:  10.1002/0471141755.ph0709s26
Online Posting Date:  October, 2004
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The isolated perfused rat liver (IPRL) has been extensively used as an intact organ model for determination of hepatic clearance and metabolism of drugs. The IPRL model can also be applied to determine physiologically based pharmacokinetics. Since the IPRL model avoids neural and hormonal interferences and excludes influences from absorption processes and non‐hepatic elimination routes such as renal excretion and respiration, it provides a relatively clean hepatic system to study metabolism and pharmacokinetics. It is especially useful to model the hepatic uptake associated with plasma protein binding and transport. The viability of the liver can be evaluated based on the gross appearance, bile flow, perfusion pressure, lactate dehydrogenase release, and oxygen uptake. The protocol describes the surgical procedures for isolation of the rat liver, a hemoglobin‐free perfusion method, and application of this model for determination of hepatic uptake and clearance.

Keywords: isolated perfused rat liver; hepatic uptake; hepatic clearance; intrinsic clearance; biliary excretion; metabolism

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Table of Contents

  • Basic Protocol 1: Analysis of Hepatic Clearance Using IPRL
  • Support Protocol 1: Viability Evaluation
  • Support Protocol 2: Measuring LDH Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Analysis of Hepatic Clearance Using IPRL

  • Male Sprague Dawley rats (200 to 350 g)
  • Anesthetic agent (e.g., 64 mg/ml sodium pentobarbital)
  • 0.5% (v/v) iodine tincture
  • Krebs‐Henseleit buffer (K‐H buffer; see recipe), ice cold
  • Saline
  • 95%:5% O 2/CO 2 gas source
  • Taurocholic acid (Sigma)
  • Test compound(s)
  • 2% to 4% (w/v) bovine serum albumin (BSA) in K‐H buffer
  • Straight/blunt, 13‐cm heavy‐duty scissors
  • Sterile gauze
  • Silk sutures
  • Sharp forceps
  • Sharp, curved 5.7‐cm scissors
  • Intramedic PE tubing (PE10, 50, 100, 160, 190, 205, 240, and 280; Thomas Scientific)
  • 60‐ml syringe
  • Medium, fine‐tipped surgical scissors
  • Perfusion setup with the following (see Fig. ):
    • Perfusion block and accessories (Accu‐Tool Corporation)
    • Techne immersion circulator (Cole‐Parmer)
    • PVC‐coated stainless tank (Cole‐Parmer)
    • Masterflex L/S digital flow monitering drive pump (Cole‐Parmer)
    • Masterflex standard pump heads (tubing size 16, LEXAN polycarbonate housing, stainless‐steel rotor; Cole‐Parmer)
    • Multiple pump head mounting hardware (Cole‐Parmer)
    • Masterflex L/S thin‐walled tubing (silicone for size 16 pump head; Cole‐Parmer)
    • Clear plastic tubing (5/16‐in. i.d. × 7/16‐in. o.d.; 1/8‐in. i.d. × 1/4‐in. o.d.; 3/32‐in. i.d. × 5/32‐in. o.d.; Baxter Scientific)
    • Sialastic medical grade tubing (0.062‐in. i.d. × 0.095‐in. o.d.)
    • 3‐way stopper (Thomas Scientific)
    • Gas dispersion tubes (Thomas Scientific)
    • Syringe pump (Harvard Apparatus)
  • 2‐liter aspirator bottles (Thomas Scientific)
  • Glass aspirators
  • Oxygenator: ∼25 feet sialastic medical‐grade tubing coiled in a partially sealed container

Support Protocol 1: Viability Evaluation

  • LDH optimized kit (Sigma)
  • Spectrophotometer
  • Quartz cuvettes
  • Glass culture tubes
  • Parafilm
  • Instruments to measure oxygen uptake:
    • Dual‐channel differential 2 amplifier (Instech Laboratories)
    • Clark‐type “Thurman‐type” electrode (Instech Laboratories)
    • Electrode cable to connect electrode to amplifier (Instech Laboratories)
    • Teflon membrane kit for oxygen electrodes (Cole‐Parmer)
    • Dual‐channel chart recorder (Cole‐Parmer)
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Literature Cited

Literature Cited
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