We are setting up some hepatocyte-based MetID experiments. Our benchmark reactions are: dealkylation of 7etoxycoumarin and conjugation of 7hydroxycoumarin to glucuronide and sulfate.
Two independent incubations of 7EC and 7HC. How come we only observe conjugation? We don't have dealkylation of 7EC rat hepatocytes.
Any clue why?
Thanks.
Thanks for the answe, Jai. My question wasn't clear, sorry. We observe conjugation in 7HC incubation only. If we incubate 7EC nothing happens. No metabolites are formed. Parent peak area does not change.
The observation of Phase II metabolites from 7HC suggests your system is metabolically viable. However, if you have lost the Phase I metabolic activity but not the Phase II activity my first thoughts are whether the solvent you have dissolved your 7EC in is having an effect? Have you tried a different solvent for your 7EC or a much lower solvent concentration?
A second thought is whether your 7EC concentration is too high so you are saturating the metabolic capacity of your cells and struggling to detect minor decreases in the 7EC concentration?
The phase II metabolism of 7EC is quite rapid. I came up with a method a while back, modified from microsomal studies, to look at this. I took the supernatent and added a sodium borate solution 1:1, which breaks the conguation of 7HCS and 7GCS, so theoretically, you are looking at your total phase I metabolism. For my application, this allowed me to look at phase I activity on a plate reader. You are measuring total 7HC, which is the Phase I metabolite. I looked at it at a excitation wavelength of 370nm and an emission wavelength of 450nm.
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