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Background
The liver is the major organ for metabolism of endogenous substrates as well as exogenous drugs. There are several in vitro tools available to help researchers study the metabolic fate of drug candidates, including isolated fresh or cryopreserved hepatocytes, liver slices, and sub-cellular fractions such as liver microsomes and S9 fractions. These sub-cellular fractions are prepared from the liver via a series of homogenization and ultracentrifugation steps.
An initial lower speed centrifugation of liver homogenate at 10,000g produces the S9 fraction also known as the supernatant of this centrifugation. The S9 fraction contains all phase I and phase II enzymes. A further centrifugation of the S9 fraction at 100,000g yields the endoplasmic reticulum-derived microsomes. Microsomes are an enriched source of cytochrome P450 (CYP) and flavin monooxygenases (FMO) enzymes. Additionally, some phase II enzymes (e.g. certain uridine glucuronide transferases (UGT) isoforms and epoxide hydrolase (EH) enzymes) are present in microsomes. . Microsomes can be used to investigate UGT activity; however, microsomal membranes restrict access of UGT substrates and/or cofactors. Optimal UGT activity can be achieve by the addition of MgCl2 and a pore-forming antibiotic (i.e. alamethicin). These components allow for the efficient transfer of a glucuronide product and the co-factor, uridine 5’-diphospho-alpha-D-glucuronic acid (UDPGA) within the microsomal matrix. Individual or pooled donor microsomes can be used for metabolism-related studies. Pooled donors can represent the “average” human population or particular factors of research interest, such as age, BMI, or limited capabilities for certain CYP isoforms.
Important notes
Note: For the measurement of UGT activity, adjust final reaction volume in step 4 to include:
For questions related to this protocol, contact us at:
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For Research Use Only. Not for use in diagnostic procedures.