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Dear Collegues,
I am doing a tissue binding studies using tissue homogenate. The
drug will be spiked into tissue homogenate and incubated, then the
free fraction of the drug will be measured.I am wondering after I
obtain the tissue homogenate, should I centrifuge briefly to clean
out the chunky part, which could not be broken by my homogenizer?
Kevin
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The following message was posted to: PharmPK
Dear Kevin,
to measure the distribution of a drug in a specific tissue, we use to
dose the drug on tissue homogenate and sonicate in HTAB buffer.
For more details you could see the following article:
Klebanoff, S. J., Waltersdorph, A., M. and Rosen, H., 1984
Antimicrobial Activity of myeloperoxidase. In: Methods in Enzymology,
vol. 105: 399-403.
Hope this helps
Marco
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I totally agree with Rostam. So long as you prepare your standards
the same way as your samples, and you know what are the total drug
concentrations in your spiked standards, you are measuring total drug
concentration in your unknown samples, assuming drug protein binding
properties are the same for both standards and samples. As for the
relative affinities of the drug binding to the solid phase vs. plasma
proteins, we don't know which one is stronger or strong enough to
grab 100% drug from the other without further investigation. Also,
0.6% MeOH doesn't seem to be able to denature plasma protein 100%.
Therefore, I would expect that you are definitely losing some protein
bound drug to the first column wash (0.6% MeOH). But again, this
should be considered simply as an issue of extraction ratio or column
recovery, a number that is very difficult to reach 100% in reality
not only for solid phase extraction but for other extraction or
sample preparation procedures as well. That is why we use internal
standard and prepare standards in parallel with the samples.
Hope this helps.
Yinuo Pang, Ph.D.
Associate Staff Scientist
Preclinical Research
ICOS Corporation
425-415-5585
ypang.-a-.icos.com
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