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The following message was posted to: PharmPK
Dear all,
I am interesting in the in vitro drug metabolism
studies using human liver microsomes. During my search
in the literature I found that Some scientists had
used NADPH as a cofactor in the incubation mixtures,
others had used NADPH generating systems (NADP+,
MgCl2, Glucose-6-phosphate dehydrogenase and
Glucose-6-phosphate). I would like to know which is
more suitable. If I would like to incubate for 10
minutes, isn't better to use NADPH instead of using
the previous mixture to generate it, and this can
avoid the delay in NADPH formation.
How rapidly NADPH could be formed from the generating
mixture.
Could you have some references concerning the role of
NADPH in the P450 catalysed drug metabolism.
Your response is highly appreciated
=3D=3D=3D=3D=3D
Youssef Hijazi
H=F4pital neurologique et neurochirurgicale
UNITE DE PHARMACOLOGIE CLINIQUE
Service Pharmaceutique
d=E9partement de dosage de m=E9dicaments et de Pharmacocin=E9tique
B.P. Lyon Montchat 69394 Lyon Cedex 03-FRANCE
Tel: 04 72 35 72 45
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The following message was posted to: PharmPK
hi
i am also performing invitro metabolism studies in rat liver post
mitochondrial fraction. it is good to use NADPH as such but it seems to be
very costly so people normally go for the NADPH generating system. moreover,
i think if you incubate the NADPH generating solution at 37=B0C for 10-15 mi=
ns
and then use it, i think it is going to work well. u must be sure that the
incubated cofactor solution should be used within 4 hrs of incubation. like
this u may avoid the delay of NADPH generation. it is just a matter of
trying out.
with best wishes and good luck
MANISH ISSAR
DEPARTMENT OF PHARMACEUTICS
UNIVERSITYOF FLORIDA
JHMHSC, BOX 100494
GAINESVILLE-32610
=46LORIDA, USA
EMAIL: manish69.-a-.ufl.edu
PHONE:352-846-2730
=46AX:352-392-4447
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The following message was posted to: PharmPK
Yousseff:
An assay system consisting of hexokinase and glucose-6-phosphate
deydrogenase has been used for many years to measure glucose mass in blood
and other tissues [Slein, M., in Methods of Enzymatic Analysis ed.
Bergmeyer, H. p.117-123 (1965)]. The formation of NADPH which is monitored
at a wavelength of 340 nanometer is stoichiometrically equivalent to the
amount of glucose originally present in solution. Final optical densities
are read 20 minutes after addition of enzymes.
I would determine the amount of NADPH cofactor which constitutes
"excess" in the microsomal prep and then compare the reaction velocity and
duration of reaction obtained with cofactor, to the reaction rate and
duration observed with NADPH generating enzyme system. If it is practical
to use the cofactor by itself then I would opt for the simpler alternative.
One other caveat . . . NADPH has a substantial extinction coefficient in
the ultraviolet spectrum which might interfere with your measurement of
microsomal activity (ie substrate-cyt P450
interaction); this could be controlled by obtaining difference spectra.
Howard S Mehler
Howard S Mehler PhD JD & Associates Inc.
FAX: (310) 271-0167
hmehler.-at-.mehler.com
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