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Dear Group,
I am studying the metabolism of a herbal compound for which a standard
is available. But there is no standard for the demetylated metabolite.
The in vitro P450 enzyme reaction is linear and I can get Km value for
the demethylation. I only can get the chromatographic peak area for
product formation. How can I get Vmax without knowing the product
concentration? Synthesizing the metabolite standard is beyond my means
and expertise.
All suggestions, comments and tips are gladly welcomed.
Chandrani Gunaratna
Chandrani Gunaratna, Ph.D.
Senior Research Scientist
Bioanalytical Systems
2701 Kent Avenue
West Lafayette, IN 47906
(765)463-4527
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Chandrani,
I recently had a similarr problem with a project for my 3rd year BSc
students -it was beyond the scope of the project finance to buy the
metabolite, but the substrate was available. Basically a collegue
suggested the following approach and it worked quite well, although it
does involve alot more work tham usual when you can meaure the
substrate. Sorry if this is too much detail ;)
Measure the substrate concentration at several time-points after the
start of the reaction -you need to accurately know the times, and they
should start very soon after the reaction is started, and continue for
a resonable time. Do this for a broad range of substrate
concentrations, just as you would for a "normal" kinetic experiment.
Then you can model the remaining substrate concentration either using
and exponential decay [Ct=Co*(exp(-k*t), where Ct is the remaining
substrate concentration, Co is the initial substrate concentration, k
is a rate constant and t is time] or buy LN transforming the data and
fitting a linear regression -the slope will be (-k) in this case. Do
this for each initial substrate concentration, but you will need to
check your data to remove the later time-points that may be beginning
to show non-linearity as you are only interested in the initial rate of
substrate loss. From this you can calcualte the "initial rate of loss
of the substrate" -in other words the initial reaction velocity (Vi),
by using k and Co:
Vi=(Co-Ct)/t where Ct is the concentration at time t, and t is very
small so as to be very soon after to the start of the reaction (so Vi
really is the initial reaction rate). Set t to be say 0.1 min, and
then using the equation above to calculate Ct=Co*(exp(-k*t). Then you
can just fit the Vi for each initial substrate concentration versus the
initail substrat oncentration normally using MM enzyme kinetics to get
Vmax and Km.
Of course this assumes that the substrate is only metabolised to a
single metabolite etc...but it worked fine for us and can be done
alomst entirely using Excel. Feel free to email me privately if you
have any questions.
David
David Foster, PhD
Department of Clinical and Experimental Pharmacology
Faculty of Health Sciences
Adelaide University
Adelaide, South Australia 5005
Email: david.foster.-at-.adelaide.edu.au
http://www.adelaide.edu.au/Pharm/index.htm
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Ask to Dr. Ehrenstorfer GmbH
Bgm. Schlosser Str.6a - D-86199 Augsburg
Fax to him the metabolite formula and ask for the standard.
Dott.ssa Paola Anfossi
Dipartimento di Sanit‡ Pubblica Veterinaria e Patologia Animale
Servizio di Prova Farmacologia e Tossicologia
http://erclib.vet.unibo.it/dspvpa/
Via Tolara di Sopra, 50 - 40064 OZZANO EMILIA (BO) - Italy
* (+39-51)792713 (centralino 2002) * (+39-51)792039
e-mail: anfossi.at.vet.unibo.it
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Dear all,
Just thinking about my reply to the MM kinetics problem that Chandrani
had, and what would happen if there are more than 1 metabolite...
I guess that the proposed method will work as long as "other"
metabolites are very minor. I suppose that Eadie-Hofstee plots of V
versus V/S could tell you if there are multiple rates of metabolism -
due to either different enzymes with different Km's etc, or the
formation (and hence loss of substrate) of different metabolites?
Unfortunaley using the loss of substrate it is not possible to tell
which proces is happening? If the Eadie-Hofstee plots suggest
> 1 enzyme, would fitting MM kinetic equations for multiple
enzymes work?
Thanks,
David Foster, PhD
Department of Clinical and Experimental Pharmacology
Faculty of Health Sciences
Adelaide University
Adelaide, South Australia 5005
Email: david.foster.-a-.adelaide.edu.au
http://www.adelaide.edu.au/Pharm/index.htm
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David, Chandrani
with the experimental procedure you proposed, (that is
measuring at known times the susbstrate disappearance
at several initial substrate concentrations,)
I do not see the need of calculating initial velocity
rates.
If you have available a non-linear regression software
able to deal with differential equations (i.e
winnonlin) you can use directly the concentration
versus time data and fit the michaelis menten equation
(that is no other thing that a differential velocity
equation). In that case you do not need to remove any
later points as you are not longer assuming linearity
(but using the M-M curve).
If you are win-nonlin users I can send you both an
example of calculations using excel and winnonlin.I'm
not an expert on metabolism but the procedure I'm
describing you is the one we have been using for
several years to characterize kinetic transport
parameters (so in our experiments we have a substrate
dissapearing by active transport)
If you suspect the involvement of a second enzyme you
havee simply to addd the second term and compare both
fits with the classical comparison procedures.
hope this helps
Marival Bermejo
Associate Professor of Pharmaceutics
Univerrsity of Valencia
Spain
http:www.uv.es/~mbermejo/absorption.htm
e mail: mbermejo.aaa.uv.es
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Dear Dr. Foster:
I have read your comments and i agree with you regarding the approach
you have described. However, this approach has to be used with lot of
caution. As you have pointed out there has to be some basic assmuptions
made prior to using this approach: the drug is metabolized by one
enzyme and only one metabolite is formed.
As you know, this type of situation is observed less frequently in
metabolism. There was a consensus report published in Pharmaceutical
Research 18(8), 2001, 1071-1080. In this report, the authors have made
comments on the product formation and substrate disappearnce approach.
I personally have some reservations using this approach, when the drug
has very low intrinsic clearance. In such case if the metabolite formed
is say 1-5% then you are looking at loss of substrate of 95-99% and the
analytical instrumentation should have sufficient precision to
distinguish between, say 99 and 98%. Such approach would be met with
more skepticsm when we study inhibiton potentials.
I am not sure if there is any study reported comparing kinetic
constants calculated form loss of substrate and product formation
approach. But, it would be a interesting comparison.
I would like to know your comments on using the substrate itself
metabolite surrogate and use it for a calibration curve. Obviously,
there will be some issues with ionization, molar absoptivity and
quantum yield differences between substrate and metabolite based on
what type of detection system is used. I would also appreciate if you
could let me know, how were your results.
Regards,
Azher M. Hussain, Ph.D.
Post Doctoral Researcher
P450 Inhibition
XenoTech, LLC
ahussain.-a-.xenotechllc.com
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All,
You know the Km, the reaction is lineair. Just use CLint=Vmax/Km ->
Vmax=Km*CLint.
If the reaction follows classical michaelis-menten kinetics and there's
only
one metabolite, this works fine.
CLint is easily determined by a single experiment at a low substrate
concentration (below Km) by :
CLint = V * ln (Cstart/Cend)/incubation period.
where V is the incubation volume, C is concentration of the substrate.
You can choose different incubation periods and different starting
concentrations (as long as they are lower than Km): the outcome should
be
the same. Choose something so that Cend is about 20% of Cstart.
(no fancy fitting programs needed, a calculator will do)
ciao,
Ruben de Kanter
Global Drug Metabolism
Pharmacia Corporation
Viale Pasteur, 10
20014 Nerviano
Italy
e-mail: ruben.dekanter.aaa.pharmacia.com
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