- On 19 Oct 2004 at 13:06:04, vuppugalla ragini (vragini.-a-.yahoo.com) sent the message

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Hi All

I have a question regarding the calculation of kinetic parameters like

Km's and Vmax's, when the drug follows either a biphasic or sigmoidal

kinetics in microsomes (assessed by Eadie-Hofstee plots). Is there an

equation to fit the observed data points in an Eadie-Hofstee plot, when

the drug displays either a biphasic pattern or sigmoidicity?. If so,

how to comeup with the input parameters (estimates) that are initially

used for fitting the observed data points?. Although I know of using

Hills and other equations for the v Vs S plots, I would like to

specifically know if there is anything like that for v Vs v/S plots

(Eadie-Hofstee). I would appreciate your help in this regard.

Thanks in advance

Ragini Vuppugalla

Gradaute Student

Texas Tech School of Pharmacy - On 19 Oct 2004 at 18:22:13, "Kavanagh, Ronald E" (KAVANAGHR.aaa.cder.fda.gov) sent the message

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A biphasic pattern might indicate 2 different enzymes.

Look at Enzyme Kinetics by Irwin H. Segel, 1975, from John Wiley &

Sons. It

will probably have what you're looking for.

Ron Kavanagh - On 20 Oct 2004 at 10:32:16, (Frederic.Massiere.at.aventis.com) sent the message

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Dear Vuppugalla,

There is a paper reviewing the aspects of atypic kinetic profiles in

microsomal metabolic reactions: J.M. Hutzler and T.S. Tracy, Drug

Metabolism and Disposition 30:355-362, 2002.

In my understanding of your question, you have: 1) used the

Eadie-Hofstee plot to visualize the occurrence of atypical kinetics, 2)

determined the equation to use for data fitting, and then 3) visualized

this equation in the V vs S plot. Then, you'd like to visualize the

curve corresponding to that equation in the Eadie-Hofstee plot,

wouldn't you ? I would try to see how a data fitting software such as

GraFit deals with that issue. There is a forum about the applications

of this software at www.erithacus.com, maybe you'll find there some

interesting information.

Hope this helps. Please let me(us) know eventually.

Frederic MASSIERE, Ph.D.

frederic.massiere.-a-.oroxcell.com - On 20 Oct 2004 at 06:47:00, "Rix, Peter" (PRix.aaa.ligand.com) sent the message
Dear Ragini,

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Why would you want to fit the data points in an Eadie-Hofstee plot? The

E-D plot, as well as the L-B and other such plots are best used only for

visual analysis of data. These days, kinetic analysis is done directly

on the V vs. S plot using nonlinear regression software. I use

SigmaPlot, but there are dozens of applications that can do this. You

can estimate some initial parameters by visual inspection of the E-H and

L-B plots.

A two-phase E-D plot usually means you have two enzymes involved, while

a sigmoidal v vs. S plot indicates allosteric interactions. Try fitting

with the Michaelis-Menten 2 enzyme or Hill equations, respectively.

Peter J Rix

Drug Safety and Disposition

Ligand Pharmaceuticals, Inc.

San Diego, CA 92121

Email: PRix.at.ligand.com - On 20 Oct 2004 at 11:59:34, vuppugalla ragini (vragini.-a-.yahoo.com) sent the message

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The reason I want to fit the data points in an Eadie-Hofstee plot is

because we can visually assess the goodness of the fit, when the drug

displays either a biphasic or sigmoidal pattern . Whereas, if I fit the

data points that are clearly biphasic on E-D plot using nonlinear

regression, I will still get a good fit (Using AIC and Schwartz

criteria) even if I use a simple michaelis-menten equation (one

enzyme). The other thing is, I agree with you in saying that we can get

some estimates of the initial parameters from the Eadie-Hofstee plot if

the drug follows a biphasic pattern, but what if the drug follows

sigmoidicity?. When I was trying to fit my data (showing sigmoidicity)

using different initial parameters I am getting very different final

parameters. In that case, which fitting should I really trust. I would

appreciate your response in this regard.

Thank you

Ragini Vuppugalla

Graduate Student

Texas Tech School of Pharmacy - On 20 Oct 2004 at 16:48:47, "Rix, Peter" (PRix.at.ligand.com) sent the message

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Ragini,

Goodness of fit can be adequately assessed after direct non linear

regression of the v vs. S plot (saturation plot). I don't think you

benefit in this respect by trying to use the E-H or L-B plot. Of

course, one does benefit by using the shape of the E-H plot as a "guide"

as to which model to attempt to fit.

Beside the obvious minimization of both the R^2 and the standard errors

of the predicted parameters, the most important criteria one should look

for is a random distribution of residuals around zero when assessing the

model selected. This is where you will hopefully see a clear skewing

when trying to fit a one enzyme MM model to data better described by a 2

enzyme MM or Hill model. If this does not produce an obvious 'winner',

there are many more exotic criteria that have been developed for this

purpose, but I must admit that I am not a modeling expert, and others

may be more qualified to comment. I would caution that analysis of

covariance may not be a good discrimination tool because in

Michaelis-Menten models, Vmax and Km share a common rate constant in

their derivations, and are by definition, intrinsically correlated.

As to getting very different final parameters based on different initial

parameters, you may find that only one of the predicted set of

parameters passes a "reality check" and the others are often nonsense

solutions.

Peter J Rix

Ligand Pharmaceuticals, Inc.

San Diego, CA

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