glucuronic acid (UDPGA) for glucuronidation, adenosine 3
0
-phosphate 50- phosphosulfate (APPS) for sulfation, glutathione (GSH) for glutathione
conjugation, and acetyl CoA for N-acetylation.
Some of these potential factors will be further discussed when appropriate.
Typical Assay Procedures The experimental procedures for determining CYP-
and UGT-mediated metabolic stability are provided. These procedures, mainly
for microsomal assays, should be amended according to the aims of the specific
study and particularly thein vitrosystem in use.
i. NADPH-dependent CYP-mediated metabolism: In a test tube
(12 75 mm), 25mL of the reaction buffer stock (10 Tris–HCl buffer)
is mixed into 170mL of the distilled and deionized (d.d.) H 2 O, followed
by the addition of 25mL of prethawed pooled HLM (20 mg/mL)
obtained from a commercial source. After 5mL of the substrate stock
solution is introduced, the test tube is pre-incubated at 37C for 1 min in
a gently shaking water bath. The reaction is initiated by the addition of
25 mL of freshly prepared NADPH solution (20 mg/mL). The protein
content can be adjusted by varying the quantity of HLM. For example,
if the final protein concentration required is 0.4 mg/mL instead of 2 mg/
mL, 5mL of HLM should be added, along with 190mL of water to make
up 195mL total volume. The final concentration of substrate is 50 times
diluted from the stock. Test tubes during the incubation should be kept
open to the air, the source of oxygen required for the reactions.
ii. UGT-mediated (or UDPGA-dependent) metabolism: In a test tube
(12 75 mm), 25mL of the reaction buffer stock (10 Tris–HCl buffer)
is mixed into 140mL of d.d. H 2 O, followed by the addition of 25mLof
prethawed pooled HLM or HLS9 (20 mg/mL) obtained commercially.
After the addition of 5mL of the substrate stock, 30mL of the mixture of
alamethicin (5mL of a 10 mg/mL solution) and saccharic acid-1,
4-lactone (25mL of a 500 mM solution) is added into the reaction
mixture. After the test tube is preincubated at 37C for 1 min in a gently
shaking waterbath, the reaction is initiated by the introduction of 25mL
of freshly prepared UDPGA (5 mM). The concentration of the proteins
can be adjusted by varying the volume of the subcellular liver
preparations. Glucuronidation by the subcellular fractions tends to be
alleviated, and thus the reaction might often require to be optimized
with the adjustments of the incubation condition such as pH.
iii. Sample clean up: At each of a set of appropriate incubation time points
(e.g., 0, 5, 15, 30, 45, 60, and 90 min), the reaction is terminated by
quickly cooling the test tube on ice, followed by the immediate addition
of an equal volume (250mL) of ice-cold methanol containing an internal
standard. The mixture is transferred into an Eppendorf tube (1.5 mL)
after being vortexed. The tube is centrifuged for 5 min in a desktop418 DETERMINATION OF METABOLIC RATES AND ENZYME KINETICS