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Dear all,
I have a question regarding some of the constituents that are required
for performing the metabolic study.
I looked in Sigma Aldrich and Fisher scientific for the components of
NADPH regenerating sytem: Glucose-6-phosphate and Glucose-6-
phosphate dehydrogenase. The following constituents are available from
these vendors.
1) For Glucose-6-phosphate: glucose-6-phosphate dipotassium salt
hydrate, glucose-6-phosphate potassium salt hydrate, glucose-6-
phosphate disodium salt hydrate, glucose-6-phosphate sodium salt
hydrate and Glucose-6-phosphate barium salt hydrate. I would like to
know which one would be efficient for the regenerating system.
2) The vendors have Glucose-6-phosphate dehydrogenase from yeast and
Leuconostoc mesenteroides. Which is the better one for NADPH
regenerating system?
It would be helpful if someone working in this area could provide
some inputs regarding the above concerns.
Thanks,
Praneeth
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Praneeth,
consistency of the ion content with your carrying buffer, using the
sodium and potassium salts would be a better circumstance if you are
using a Na/K salt based buffering system. Stay away from the barium
salts as they are not routinely used in metabolic stability
experiments and I do not believe that anyone has examined the presence
of barium in incubation mixtures on reaction rates, as well as NADPH
generation. Use the Leuconostoc mesenteroides version of the
dehydrogenase. I would also recommend that you test the generation of
your NADPH by creating a mock incubation and monitoring the kinetic
formation of NADPH at 340 nm, if possible. This will give you an idea
of the total carrying capacity of your system for the generation of
NADPH. There are numerous publications in DMD as well as several good
text books ( Fundamentals of drug metabolism and drug disposition. /
Edited by Bert N. La Du, H. George Mandel [and] E. Leong Way) that
contain excellent recipe-based cocktails.
Glucose 6- Phosphate Sigma G7879
NADP+ Sigma N0505
MgCl2 Prepared fresh
Glucose-6-Phosphate dehydrogenase Sigma G7877
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PharmPK - Discussions about Pharmacokinetics
Pharmacodynamics and related topics
Reagents:
- 20 x NADPH-generating system: NADP (20 mM), glucose-6-phosphate (100
mM), and glucose-6-phosphate dehydrogenase (20 Unit/mL).
Q1: what solvent do you to prepare these 3 components
A:1 Use phosphate buffer or the buffer you are using for the
experiment.
Q2: Can you prepare stocks of these 3 components and store them? Are
they stable? Or do you prepare and premix them immediately before
every experiment?
A2: No, the NRS solution should be prepared fresh. Do not vortex
after addition of enzyme as it may denature it.
Q3: How long should you weight before using the system to allow enough
time for NADPH generation?
A3: Allow it for thawing for 10-15 min and should be used within 1
hour of preparation.
Q4: Can you store and re-use (any stability issues)?
- 20x human microsomes (20 mg/ml) in 50 mM KH2PO4 pH7.4 buffer
containing 3mM MgCl2.
-20 x test compound (20 uM) in MeOH
A4: Microsomal suspension should always be prepared fresh amd the
stock in methanol may evaporate even at refrigerated temperature so
better to prepare in Acetonitrile or prepare fresh working stocks.
(organic content should be as low as possible <2%)
The microsomal suspension should be of 0.5 to 1 mg/mL of protein
concentration.
The test compound should be less than Km or as low as possible (based
on lower limit of quantification of respective analytical method)
Also put a control experiment without the addition of cofactors
(sampling at 0, 30, 60 and 120 min) to check any non-microsomal
degradation.
We have used glucose-6-phosphate disodium salt hydrate and Glucose-6-
phosphate dehydrogenase from Leuconostoc mesenteroides both worked well.
Simarjeet Kaur
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The following message was posted to: PharmPK
Praneeth,
In general barium salts are much less soluble vis-a-vis sodium and
potassium salts. One does not need additional inaccuracies in such
assays.
Ving J. Lee
CEO-CSO
Adesis Inc.
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