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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Analysis of Hepatic Clearance Using IPRL

  Materials
  • 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

  Materials
  • 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)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Anundi, I., Kauffman, F.C., el‐Mouelhi, M., and Thurman, R.G. 1987. Hydrolysis of 4‐methylumbelliferyl sulfate in periportal and pericentral areas of the liver lobule. Arch. Toxicol. 60:69‐72.
   Bernard, C. 1855. Sur le mecanism de la formation du sucre dans le foie, C. R. Acad. Sci. 41:461‐469.
   Booth, C.L., Pollack, G.M., and Brouwer, K.L. 1996. Hepatobiliary disposition of valproic acid and valproate glucuronide: Use of a pharmacokinetic model to examine the rate‐limiting steps and potential sites of drug interactions. Hepatology 23:771‐780.
   Brouwer, K.L. 1997. Hepatic distribution and clearance of antisense oligonucleotides in the isolated perfused rat liver. Pharm. Res. 14:516‐521.
   Brouwer, K.L. and Thurman, R.G. 1996. Isolated perfused liver. In Models for Assessing Drug Absorption and Metabolism (R.T. Borchardt, P.L. Smith, and G. Wilson, eds.) Plenum Press, New York.
   Brouwer, K.L.R. and Vore, M. 1985. The effect of hypoxia and pregnancy on antipyrine metabolism in isolated perfused rat liver. J. Pharmacol. Exp. Ther. 234:584‐589.
   Conway, J.G., Kauffman, F.C., Tskuda, T., and Thurman, R.G. 1987. Glucuronidation of 7‐hydroxycoumarin in periportal and pericentral regions of the lobule in livers from untreated and 3‐methylcholanthrene‐treated rats. Mol. Pharmacol. 33:111‐119.
   Durham, S. and Vore, M. 1985. Taurocholate and steroid glucuronides. Mutual protection against cholestasis in the isolated perfused rat liver. J. Pharmacol. Exp. Ther. 237:490‐495.
   Foy, D.B., Toxopeus, C., and Frazier, M.J. 1999. Kinetic modeling of slow dissociation of bromosulphophthalein from albumin in perfused rat liver: Toxicology implications. Toxicol. Sciences 50:20‐29.
   Frazier, M.J. 1998. Biological based kinetic model for chemical kinetics in the isolated perfused rat liver system: Basic recirculation model for water‐soluble/lipid‐insoluble chemicals (Log Po/w<−1). Toxicol. Methods 8:257‐284.
   Ganey, P.E., Kauffman, F.C., and Thurman, R.G. 1988. Oxygen‐dependent hepatotoxicity due to doxorubicin: Role of reducing equivalent supply in perfused rat liver. Mol. Pharmacol. 34:695‐701.
   Kari, F.W., Kauffman, F.C., and Thurman, R.G. 1985. Effect of bile salts on rates of formation, accumulation, and export of mutagenic metabolites from benzo(a)pyrene produced by the perfused rat liver. Cancer Res. 45:1621‐1627.
   Liu, Y. and Thurman, G.R. 1992. Potentiation of adriamycin toxicity by ethanol in perfused rat liver. J. Pharmacol. Exp. Ther. 263:651‐656.
   Liu, Y., Hyde, F.J., and Vore, M. 1992. Prolactin regulates maternal bile secretory function post partum. J. Pharmacol. Exp. Ther. 261:560‐566.
   Liu, Y., Huang, L., Hoffman, T., Gosland, M., and Vore, M. 1996. MDR1 substrates/modulators protect against estrodiol 17 β‐(β‐D‐glucuronide) cholestasis in the rat liver. Cancer Res. 56:4992‐4997.
   Liu, Y., Bacon, E.R., Ballinger, K., Black, C.D.V., Illig, K., Mcintire, G.L., Wang, P.P., O'Neil, N., Kinter, L., and Desai, V.C. 2000. Pharmacokinetics and hepatic disposition of bis[1‐(ethoxycarbonyl)propyl]5‐acetylamino‐2,4,6‐triiodoisophthalate in rats and isolated perfused rat livers. Drug Metab. Dispos. 28:731‐736.
   Miller, L.L., Bly, C.G., Watson, M.L., and Bale, W.F. 1951. The dominant role of the liver in plasma protein synthesis: Direct study of isolated perfused rat liver with aid of lysine‐ν‐C14. J. Exp. Med. 94:431‐453.
   Nolting, A., DeLong, R.K., Fisher, M.H., Wickstrom, E., Pollack, G.M., Juliano, R.L., Pang, K.S., and Rowland, M. 1977. Hepatic clearance of drugs, II. Experimental evidence for acceptance of the “well‐stirred” model over the “parallel tube” model using lidocaine in the perfused rat liver in situ preparation, J. Pharmacokinet. Biopharm. 5:655‐680.
   Proost, J.H., Nijssen, H.M.J., Strating, C.B., Meijer, D.K.F., and Groothuis, G.M. 1993. Pharmacokinetic modeling of the sinusoidal efflux of anionic ligands from the isolated perfused rat liver: The influence of albumin, J. Pharmacokinet. Biopharm. 21:375‐394.
   Scholz, R. and Bücher, T. 1965. Haemoglobin‐free perfusion of rat liver. In Control of Energy Metabolism (B. Chance, ed.), pp. 393‐414. Academic Press, New York.
   Thurman, R.G., Kauffman, F.C., and Jungerman, K. 1986. Regulation of Hepatic Metabolism: Intra‐ and Intercellular Compartmentation, Plenum Press, N.Y.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library