w Single substrate
concentration activity
w Scaling factor
w Western blot
w Enzyme kinetics
Corning
®
UltraPool
™
HLM 150
Characterization
Enzyme Measured Assay Specic Content or Activity
Total P450 Omura and Sato 350 pmol/mg
OR Cytochrome c Reductase 270 nmol/min/mg
Cytochrome b5 Spectrophotometric 660 pmol/mg
CYP Isoform Probe Substrate Activity [S] (μM) Activity (pmol/min/mg)
CYP1A2 Phenacetin O-deethylase 100 650
CYP2A6 Coumarin 7-hydroxylase 100 1000
CYP2B6 (S)-Mephenytoin N-demethylase 100 54
CYP2C8
Paclitaxel 6a-hydroxylase
100 260
CYP2C9 Diclofenac 4'-hydroxylase 100 2900
CYP2C19 (S)-Mephenytoin 4'-hydroxylase 100 87
CYP2D6
Bufuralol 1'-hydroxylase (amount of
activity inhibited by 1 µM quinidine)
25 75
CYP2E1 Chlorzoxazone 6-hydroxylase 1000 3600
CYP3A4
Testosterone 6b-hydroxylase
200 5300
CYP4A11 Lauric acid 12-hydroxylase 100 1500
FMO Methyl p-Tolyl Sulfide Oxidase 1000 1300
UGT1A1 Estradiol 3-Glucuronidation 100 930
UGT1A4 Trifluoperazine N-Glucuronidation 100 850
UGT1A6 Serotonin Glucuronidation 1000 16000
UGT1A9 Propofol Glucuronidation 20 2000
UGT2B7 AZT Glucuronidation 1000 720
Methods
w All cytochrome P450 assays conducted at 0.8 mg/mL protein (except CYP3A4, which was at 0.5 mg/mL)
with an NADPH generating system (1.3 mM NADP, 3.3 mM glucose 6-phosphate and 0.4 U/mL glucose
6-phosphate dehydrogenase), 3.3 mM MgCl
2
, and incubated for 20 minutes or 10 minutes (CYP2C8,
CYP2C9, CYP3A4 and CYP4A).
w 0.1 M Potassium phosphate buffer (pH 7.4) was used for all P450 enzymes except CYP2B6, CYP2C8, and
CYP2C19 (0.05 M) and CYP2A6, CYP2C9, and CYP4A which used 0.1 M Tris (pH 7.5).
w The FMO assay was conducted in the same volume and protein concentration in 0.05 M glycine buffer
(pH 9.5) with the same NADPH generating system, 3.3 mM MgCl
2
, 1.2 mM diethylenetriaminepentacetic
acid, 0.5 mg/mL Triton X-100 and incubated for 10 minutes.
w UGT Glucuronidation assays contained 0.5 mg/mL protein for UGT1A1 and 1A4, 0.1 mg/mL for 1A6, 0.15
mg/mL for 1A9 and 0.8 mg/mL for 2B7, along with 2 mM UDPGA, 10 mM MgCl
2
, 25 µg/mL Alamethicin
in 50 mM Tris-HCl buffer (pH 7.5).
w UGT1A1 was incubated for 30 minutes, 1A4 for 20 minutes, 1A6 for 15 minutes, 1A9 for 10 minutes and
2B7 for 25 minutes.
Microsome Scaling Factor in 150 Donor Pool
mg Microsomal Protein/gram Liver
43
Methods
Scaling factor was determined by the Matsubara method (Anal. Biochem., Vol. 75, p. 596, 1976).
CYP Isoform Abundance in 150 Donor Pool
Enzyme Measured Assay CYP Abundance (pmol/mg)
CYP3A4 Western Blot 71
CYP3A5 Western Blot 10
Methods
w The Western Blot assay was carried out using standard protocols. SDS-gel electrophoresis was by the method of
Laemmli (Laemmli, U.K, 1970, Nature, 227: 680-685).
w CYP protein abundance in the pooled HLM was quantitated using authentic standards derived from recombinant
P450 isoforms and purified proteins.
Kinetic Constants for CYP Isoform Activities for Corning UltraPool HLM 150
Enzyme
Measured Substrate
Assay Protein
Concentration
(mg/mL)
Assay Time
(min) [S] Range Km (μM)
Vmax
(pmol/min/mg)
CYP1A2 Phenacetin 0.2 10 2.5-150 µM 24 1006
CYP2A6 Coumarin 0.05 5 0.1-20 µM 1.0 471
CYP2B6 Bupropion 0.1 5 15-300 µM 137 302
CYP2C8 Amodiaquine 0.02 5 0.125-10 µM 0.90 2023
CYP2C9 Diclofenac 0.05 5 0.69-40 µM 3.5 3973
CYP2C19 (S)-Mephenytoin 0.3 10 5-200 µM 24 53
CYP2D6 Dextromethorphan 0.1 5 0.5-50 µM 5.0 246
CYP2E1 Chlorzoxazone 0.1 5 15-500 µM 68 2311
CYP3A4 Midazolam 0.02 5 0.25-20 µM 1.8 1527
CYP3A4 Testosterone 0.05 10 5.0-250 µM 64 (n=1.2) 5086
Methods
w Incubations for Km and Vmax determination contained 100 mM Phosphate (pH 7.4), 3.3 mM MgCl
2
, NADPH
gener ating system (1.3 mM NADP, 3.3 mM glucose 6-phosphate and 0.4 U/mL glucose 6-phosphate dehydrogenase)
and CYP probe substrate (10 to 12 concentrations evenly spaced over the range).
w Metabolite formation was analyzed using validated LC-MS/MS methods with stable isotope-labeled metabolites as
internal standards.
w For testosterone, the Km column represented as S50 (Hill coefficient = 1.2).
Corning
®
UltraPool
™
HLM 150 Characterization