Michael Court
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June 2001
UGT activity by HPLC – method development
Develop HPLC method to measure glucuronide levels
a. Check for previous method references
b. Dissolve substrate and glucuronide in methanol (10 mg / 100 mL)
c. Dry down substrate and glucuronide and reconstitute in 50 mM PO4 buffer (substrate
concentration is Km value and 10x Km)
d. Run UV (or fluorescent) scan of substrate and glucuronide and determine the lambda
max for the analyte – set detector to this value
e. Set up HPLC to equilibrate
i. 20 mM KH2PO4 pH = 4.5 (uncorrected pH)
ii. start at 0% acetonitrile concentration
iii. flow rate 1 mL/minute (pressure < 200 bar)
f. Inject ~10 uL of sample
g. Run acetonitrile gradient from 0% to 50% over 30 minutes (return to 0% at end)
h. Optimize method (starting and end acetonitrile concentration) such that you get
separation of glucuronide and other peaks and substrate in reasonable time (<40
minutes; ideally 20 minutes)
i. Identify a suitable internal standard – one that separates well from substrate and product
and ideally something chemically similar to substrate or product
j. Check this method using a liver microsome incubation (incubate substrate with
UDPGA and microsomes for 0 minutes and 60 minutes)
k. If no glucuronide standard – identify potential glucuronide peak (compare 0 min and 60
minute incubation samples) and treat 60 minute sample with beta-glucuronidase
(Glusulase) – glucuronide should disappear.
Time linearity study at fixed liver microsome protein concentration (0.8 mg/mL protein –
establish highest time that can be used with linear product formation. Eg. 2.5, 5, 10, 15, 20,
30, 45, 60 minutes
Protein linearity study using the time from previous study. Eg. 0.1, 0.2, 0.3, 0.4, 0.6, 0.8,
1.0 mg/mL
Alamethicin activation study using time and protein from previous study.
a. Alamethicin stock solution = 2.5 mg / mL (in methanol)
b. Final concentration = 0.025 mg / mL (add 2.5 uL per 250 uL incubation volume) for
0.8 mg / mL protein concentration
c. 0.03125 mg alamethicin per mg protein
d. Try 0.01, 0.02, 0.03, 0.05, 0.1, 0.2 mg alamethicin per mg protein
See if you can find Km for liver microsomes with this substrate. Do a Km study if has not
been done before (or if not done well before).
a. Eg. 1, 2, 5, 10, 20, 50, 100 uM (if Km = 10 uM)
b. Need to have 2 – 3 points below final Km and up to 10x Km
c. Generally use 8-10 points in duplicate
Activities for all recombinant UGTs at substrate Km and 10x Km (UDPGA = 5 mM;
MgCl2 = 5mM). Protein 0.5 mg / mL. In duplicate if 100 uL incubation volume; otherwise
do only single assay per UGT.
Activities for liver microsomes, which have UGT1A6 protein measurements at either Km
OR 10x Km substrate concentration whichever concentration shows highest activity with
UGT1A6 relative to other isoforms.