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Microsomes, S9 Fractions and Cytosol

Your metabolic tools for drug discovery & development
CellzDirect™ microsomes & other liver subcellular fractions

  • Large pooled lots for reproducible, long-term studies
  • Fractions carefully isolated by experts in hepatic biology
  • Microsomes functionally tested for select CYP450 and Phase II enzyme activities

Human microsome pools and subcellular fractions

Subcellular fractions, derived from the endoplasmic reticulum of liver, contain a variety of metabolic enzymes for assessing the in vitro metabolism of drug candidates (Table 1) and are suitable for a variety of experiments:

  • Cytochrome P450 inhibition studies
  • Metabolic stability
  • Cytochrome P450 phenotyping
  • Metabolite characterization

Invitrogen™ provides liver subcellular fractions from a variety of tox species, including human.  Each product contains an average representative pool of donors.  Human microsome pools are fully characterized (Km and Vmax) according to GLP standards for major cytochrome P450 activities and select Phase II enzymes using FDA recommended substrates (Tables 2-3).

Table 1 - Relevant applications for liver subcellular fractions.


Metabolic  EnzymesLiver MicrosomesLiver S9 FractionsLiver Cytosol
Aldehyde Oxidase

X
X
Cytochromes P450 (CYP)X
X

Flavin Monooxygenases (FMO)
X
X

Glutathione Transferase (GST)
X

Monamine Oxidase (MAO)

X

Sulfurotrnasferase (SULT)

X
X
Uridine Glucuronide Transferase (UGT)
X
X

Table 2 - Cytochrome P450 and Phase II enzymes tested in CellzDirect™ human microsome pools.


Enzyme MonitoredMarker SubstrateIncubation (min)Protein Concentration (mg/ml)Metabolite
CYP1A1/2
Phenacetin
30
0.1
Acetaminophen
CYP2A6
Coumarin
5
0.025
7-Hydroxycoumarin
CYP2B6
Bupropion
20
0.25
Hydroxybupropion
CYP2C8
Paclitaxel
10
0.075
6α-Hydroxypaclitaxel
CYP2C9
Tolbutamide
20
0.1
Hydroxytolubutamide
CYP2C19
(S) Mephenytoin
30
0.1
4'-Hydroxymephenytoin
CYP2D6
Dextromethorphan
15
0.2
Dextrorphan
CYP2E1
Chlorzoxazone
20
0.1
6-Hydroxychlorzoxazone
CYP3A4/5
Midazolam4
0.025
1'-Hydroxymidazolam
CYP3A4/5
Testosterone7
0.05
6β-Hydroxytestosterone
CYP4A11
Lauric Acid
15
0.2
12-Hydroxydecanoic Acid
FMO
Methyl p-Tolylsulfide15
0.05
Methyl p-Tolylsulfide
UGT
7-Hydroxycoumarin
30
0.2
7-Hydroxycoumarin Glucuronide

Table 3 - Kinetic parameters for the CellzDirect™ 50 donor human liver microsome pool.


IsoformMetaboliteKm(μM)Vmax (nmol/min/mg)
CYP1A2Acetaminophen780.73
CYP2A67-Hydroxycoumarin1.10.53
CYP2B6Hydroxybupropion640.29
CYP2C86α-Hydroxypaclitaxel5.50.15
CYP2C9Hydroxytolbutamide2200.19
CYP2C194'-Hydroxymephenytoin340.031
CYP2D6Dextrophan3.20.13
CYP2E16-Hydroxychlorzoxazone701.4
CYP3A46β-Hydroxytestosterone194.0
CYP3A41'-Hydroxymidazolam1.61.1