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The following message was posted to: PharmPK
Dear PharmPKmembers,
with our working group, recently we started performing enzyme kinetic
studies with liver microsomes, and after reading the appropriate literature,
some questions arose:
As a very general condition in order to determine Km/Vmax/Ki, all papers say
that as a incubation time, a time should be used where product formation is
linear with respect to time and protein; e.g., between 5 and 15 min. Hmm,
strange: if the product formation is linear to time, this significates
actually that we are working in the enzyme saturation, but for determining
the Ki, one should measure at Km concentrations, and the Km is out of the
saturation range .... How can this be? But anyway: HOW can one determine the
linearity of the product formation with respect to time and protein?
Second: Is Ki, the inhibition constant, once found, substrate-dependent or
is it applicable on different substrates? We suppose the latter, assuming
that there is the same microsomal system. But this implicates that the Ki
values are not comparable to others won by other labs, due to the different
microsomal systems used in different labs!?
Thank you very much for helping us
Stefan Steinmeyer
Stefan Steinmeyer
University of Saarland
Institute of Legal Medicine, Toxicology
Building 42
D-66421 Homburg/Saar
Germany
Tel.: ++49-6841/166315
Fax.: ++49-6841/166314
E-Mail: rmsste.-at-.med-rz.uni-sb.de
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The reason it's linear is because you're using initial rate conditions.
That is the substrate concentration does not change appreciably.
For example if the conc is 10 mcg/ml and the volume is 10L, the total
amount is 1 gm. Under linear conditions (i.e. Conc is << Km)if Cl is 10
ml/min then every 5 minutes approximately another 50 mcg is eliminated,
but since it doesn't change the substate concentration measurably the
rate doesn't change by a detectable amount. This is also why you need to
measure rate of production rather than rate of elimination.
As to determining your conditions, just do incubations with different
durations and protein concentrations using a couple of concentrations.
Second question. Maybe yes and maybe no. Enzymes may have multiple
binding pockets, genotypes/phenotypes, post-translational modifications
or mechanisms of inhibition. Therefore Ki's may or may not be able to be
compared for different substrates or for different enzyme sources. In
addition incubation conditions may be different. Consequently, these are
likely ballpark estimates and you need to evaluate multiple enzyme
sources / substrates with known differences.
I recommend Segal's text on Enzyme Kinetics.
Ronald E. Kavanagh. B.S. Pharm., Pharm.D., Ph.D.
Food & Drug Administration
Office of Clinical Pharmacology and Biopharmaceutics
WOC-II Rm 4061 HFD-860
1451 Rockville Pike
Rockville, MD 20852
Phone: 301-594-6650 FAX: 301-480-3212
Office: Woodmont (WOC-II) Rm. 4061 e-mail: kavanaghr.-at-.cder.fda.gov
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The following message was posted to: PharmPK
Dear Stefan
Standard Km/Vmax estimates need to be carried out under linear
conditions. Linearity in this case means you incubate at a fixed
substrate and cofactor concentration for varying times. The product
formed (even substrate consumed) should be linear with time, this
signifies that you are in the linear portion of the substrate versus
velocity curve. You should also ensure that you are linear with
respect to protein or enzyme concentration as well. This then
justifies normalising velocity for time and protein/enzyme content.
Enzyme Kinetics, by I.H. Segel, John Wiley & Son 1975 explain this
well.
We have generally found that for competitive inhibitors the Ki of an
inhibitor against an enzyme is generally maintained across substrates
and microsomal systems. We found that quinidine had similar Ki
towards CYP2D6 in both a cumene hydroperoxide system and NADPH/O2
system (Bichara et al DMD 24:112, 1986)
Hany Ghabrial
Dr. Hany Ghabrial Telephone: (61+3) 9496 2549
Department of Medicine Fax : (61+3) 9497 4554
Austin & Repatriation Medical Centre
West Heidelberg Victoria 3081
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The following message was posted to: PharmPK
Linearity with respect to time doesn't really exist
during these initial rate type experiments. I
sometimes find it frustration when someone (like a
reviewer) asks of my experiment, "were you in the
linear range?". What linear range? Does the progress
curve suddenly become non-linear after 5 or 10
minutes? Of course not, it's always non-linear!
What you should try to do is design your experiment so
that less than 10-20% of your substrate is consumed.
Even then you will be underestimating the initial
rate... but not by much. If you want better accuracy
there are corrections you can make (see Waley (1981)
Biochem. J. 193, 1009-1012).
A few multiple time point experiments to assess
linearity would also be useful... in case of product
inhibition or low enzyme stability, for example.
A good paper on determining Ki values:
Kakkar et al (1999) Drug Metab Dispos 27, 756-762
Cheers,
Steve
Stephen Day
Merck-Frosst Centre for Therapeutic Research
Kirkland, QC CANADA
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