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Dear All,
I would be very grateful for your input/advice on liver S9 incubation
experiments.
1) What is the most appropriate buffer for a S9 experiment followed
by LC/MS analysis? I have a number of published articles detailing
experimental procedures using phosphate buffer but according to
Sigma, Glucose-6-phosphate dehydrogenase is inhibited by this
buffer. Furthermore the activities of some enzymes are higher in
tris buffer. Shouldn't Tris be the preferred option?
2) What is the purpose of MgCl2 in the NADPH re-generating system?
3) Can I use glutathione, potassium cyanide, semicarbazide as
chemical traps for reactive metabolites on S9 incubations?
Many thanks for your help
Raniero Zazzeroni
Raniero Zazzeroni
Unilever - Safety & Environmental Assurance Centre
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Dear Raniero,
1) I would use Tris buffer instead of phosphate buffer. The type of
analysis is not important - the most important issue is to get the
highest enzyme activity. But if you are using LC/MS, I would advise
you to use an extraction procedure prior to chromatography (SPE /
LLE) or use a flow-diverter valve after column to prevent the buffer
peak from reaching the MS ionization source. It may clog the capillar
or the inlet orrifice.
2) MgCl2 is important for some transferases for example UGT. Mg++ is
a cofactor.
NADPH regeneration is necessary for CYP reactions, because NADPH-CYP
reductase converts CYP's Fe+++ ion to Fe++ ion which can only then
bind O2 and change back to Fe+++.
3) I think i have read an article that reported the use of
semicarbazide as a reactive metabolite trap. Be careful about the
concentration, though, as it may inhibit your metabolizing enzyme.
Hope that helped,
Jurij T
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