Lab 7: Induction/inhibition Of Drug Metabolims In Vitro Flashcards
How was the microsomal oxidising capacity assay conducted?
Tris buffer and MgCl2 along with substrates to the enzymes were added in eppendorf tubes along with either control, cimetidine or phenol drug.
Two copies of each parameter was prepared.
Microsome samples were added to all tubes and they were subsequently incubated for 2 minutes.
NADPH was added followed by incubation for 15mins.
Methanol was added before placing all tubes on the ice bath for 10 minutes.
Tubes were centrifuged for 5 minutes,
the supernatant was pipetted out of each one and into in the spectrophotometer plate and the fluorescence measured.
What are the two assays conducted in this lab?
Microsomal oxidising capacity using EROD and PROD activity
Spectral determination of the amount of Cytochrome p450
What is ethoxyresorufin?
The substrate to EROD
What is pentoxyresorufin?
The substrate to PROD
Why was tris buffer added?
To stabilise the hydrogen ion concentrations so that they do not affect the enzyme function
Why was NADPH added?
To provide an electron source for the metabolism reaction
Why were the tubes incubated at 37°C?
This activates the enzymes
Why was methanol added to all of the tubes after the 15 minutes of imcubation?
To stop the reaction
Why is the enzyme activity detected by fluorescence?
The enzyme products (resorufin) will fluoresce at 530nm (excitation) and 585nm (emission) and can thus be used to detect the extent of enzyme activity
What are the results of the microsomal oxidising capacity assay ?
We found that phenobarbitone is preferentially metabolised by PROD
This is shown by the 3331% increased activity resulting from the PROD enzyme with pheno added.
We also found that cimetidine did inhibit metabolism as tubes containing cimetidine had lower fluorescence
While phenobarbitone did induce metabolism as tubes containing pheno had a higher fluorescence
Why was the microsomal oxidising capacity assay conducted?
EROD and PROD are natural mouse enzymes. The assay was undertaken to assess the effects of pheno and cimetidine on the enzyme activity to see if the induction and inhibition of metabolism is in fact a pharmacokinetic interaction
What is the purpose of the spectral determination of cytochrome P450 assay?
To assess the amount of CYP450 in the liver enzymes…????
Why is Cytochrome P450 being a haemoprotein significant?
When it is reduced and complexed with CO it results in a characteristic absorption spectrum primarily around 450nm
How was the spectral determination of cytochrome P450 assay undertaken?
The microsomal fraction was pipetted into the tube and placed on ice.
Glycerol phosphate buffer was added
Sodium dithionite was added and the tube was gently inverted.
CO was carefully bubbled before the sample was scanned at 440-500 nm.
The absorbance between these two values were recorded.
Why is the glycerol phosphate buffer added?
This inhibits the conversion from cytochrome p450 –> cytochrome P420
