I have conducted in vitro metabolic studies using human liver microsomes(HLM) and the incubation didn't work. I am not sure what the problem might be. The incubation mixture contained 25uM nicotine, 1mM NADPH, 5mM MgChloride, 10mM sodium pyrophosphate and 0.5mg/ml HLM. The final volume of the incubation mixture was 500uM. The incubation was terminated after 30 minutes by adding 20uM acetonitrile. This was followed by centrifugation at 14000rpm for 10 minutes and the supernatant was analyzed using HPLC.
does anyone have any idea of what might be wrong?
Did you include a buffer to fix the pH (sodium pyrophosphate?) and what pH did you choose?
I used potassium phosphate buffer (pH 7.4). I included Sodium pyrophosphate because it is an inhibitor of serine/threonine phosphatase that would otherwise degrade FMN to riboflavin and hence disrupt the NADPH-CYP450 oxidoreductase system.
what I did HPLC analysis, I did not see any new peak corresponding to a product. I am using nicotine and caffeine as a positive control. None of the two seems to have worked. However, The glucuronidation reaction conducted using microsomes from the same lot have worked out well. However, the metabolism of caffeine and nicotine have not worked.
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