Two metabolites of a new compound were identified after incubation with pooled human liver microsomes, a desmethyl metabolite and a ring hydroxylated metabolite. The new compound was incubated with samples from a human liver microsome bank, and the amount of metabolite formed was compared with activities for probe substrates. The data are available in the table on the next slide. Correlation with CYP probe substrates Metabolite CYP2A6 CYP2B6 CYP2C19 CYP2D6 CYP3A4 Desmethyl 0.22 NS 0.33 NS 0.87 *** 0.41 NS 0.52 * R-hydroxyl 0.41 * 0.79 *** 0.24 * 0.21 NS 0.44 * Data are correlation coefficients with associated P values (a) The metabolites were identified and quantified using liquid chromatography, coupled to mass spectrometry (triple quadrupole) using selected reaction monitoring. Give a brief explanation of selected reaction monitoring, including a description of how each of the quadrupole mass analysers contributes to detection. (b) What would be the appropriate units for metabolite formation and probe substrate activity for these correlation studies? (c) A significant P value (shown by the asterisks) shows that the gradient of the regression line is significantly different from what value? (d) What do the correlation data suggest about the enzyme responsible for metabolite formation? (e) What further studies would you carry out to gain further information and confirmationTwo metabolites of a new compound were identified after incubation with pooled human liver microsomes, a desmethyl metabolite and a ring hydroxylated metabolite. The new compound was incubated with samples from a human liver microsome bank, and the amount of metabolite formed was compared with activities for probe substrates. The data are available in the table on the next slide. Correlation with CYP probe substrates Metabolite CYP2A6 CYP2B6 CYP2C19 CYP2D6 CYP3A4 Desmethyl 0.22 NS 0.33 NS 0.87 *** 0.41 NS 0.52 * R-hydroxyl 0.41 * 0.79 *** 0.24 * 0.21 NS 0.44 * Data are correlation coefficients with associated P values (a) The metabolites were identified and quantified using liquid chromatography, coupled to mass spectrometry (triple quadrupole) using selected reaction monitoring. Give a brief explanation of selected reaction monitoring, including a description of how each of the quadrupole mass analysers contributes to detection. (b) What would be the appropriate units for metabolite formation and probe substrate activity for these correlation studies? (c) A significant P value (shown by the asterisks) shows that the gradient of the regression line is significantly different from what value? (d) What do the correlation data suggest about the enzyme responsible for metabolite formation? (e) What further studies would you carry out to gain further information and confirmation
Question
Two metabolites of a new compound were identified after incubation with pooled human liver microsomes, a desmethyl metabolite and a ring hydroxylated metabolite. The new compound was incubated with samples from a human liver microsome bank, and the amount of metabolite formed was compared with activities for probe substrates. The data are available in the table on the next slide. Correlation with CYP probe substrates Metabolite CYP2A6 CYP2B6 CYP2C19 CYP2D6 CYP3A4 Desmethyl 0.22 NS 0.33 NS 0.87 *** 0.41 NS 0.52 * R-hydroxyl 0.41 * 0.79 *** 0.24 * 0.21 NS 0.44 * Data are correlation coefficients with associated P values (a) The metabolites were identified and quantified using liquid chromatography, coupled to mass spectrometry (triple quadrupole) using selected reaction monitoring. Give a brief explanation of selected reaction monitoring, including a description of how each of the quadrupole mass analysers contributes to detection. (b) What would be the appropriate units for metabolite formation and probe substrate activity for these correlation studies? (c) A significant P value (shown by the asterisks) shows that the gradient of the regression line is significantly different from what value? (d) What do the correlation data suggest about the enzyme responsible for metabolite formation? (e) What further studies would you carry out to gain further information and confirmationTwo metabolites of a new compound were identified after incubation with pooled human liver microsomes, a desmethyl metabolite and a ring hydroxylated metabolite. The new compound was incubated with samples from a human liver microsome bank, and the amount of metabolite formed was compared with activities for probe substrates. The data are available in the table on the next slide. Correlation with CYP probe substrates Metabolite CYP2A6 CYP2B6 CYP2C19 CYP2D6 CYP3A4 Desmethyl 0.22 NS 0.33 NS 0.87 *** 0.41 NS 0.52 * R-hydroxyl 0.41 * 0.79 *** 0.24 * 0.21 NS 0.44 * Data are correlation coefficients with associated P values (a) The metabolites were identified and quantified using liquid chromatography, coupled to mass spectrometry (triple quadrupole) using selected reaction monitoring. Give a brief explanation of selected reaction monitoring, including a description of how each of the quadrupole mass analysers contributes to detection. (b) What would be the appropriate units for metabolite formation and probe substrate activity for these correlation studies? (c) A significant P value (shown by the asterisks) shows that the gradient of the regression line is significantly different from what value? (d) What do the correlation data suggest about the enzyme responsible for metabolite formation? (e) What further studies would you carry out to gain further information and confirmation
Solution
(a) Selected Reaction Monitoring (SRM) is a method used in mass spectrometry where a specific precursor ion is selected in the first stage of a tandem mass spectrometer and an expected product ion is selected in the second mass spectrometer stage for detection. The first quadrupole (Q1) acts as a mass filter to select the precursor ions. The second quadrupole (Q2) is a collision cell where selected precursor ions are fragmented. The third quadrupole (Q3) again acts as a mass filter to select specific fragment ions for detection.
(b) The appropriate units for metabolite formation would be concentration (such as nM, µM) over time (such as min, hr), indicating the rate of metabolite formation. The units for probe substrate activity would also be concentration over time, indicating the rate at which the substrate is metabolized.
(c) A significant P value shows that the gradient of the regression line is significantly different from zero. This indicates that there is a significant relationship between the variables being studied.
(d) The correlation data suggest that the enzyme CYP2D6 is most likely responsible for the formation of the desmethyl metabolite, as it has the highest correlation coefficient (0.87) and a significant P value. For the R-hydroxyl metabolite, the enzyme CYP2B6 seems to be most responsible, with a correlation coefficient of 0.79 and a significant P value.
(e) Further studies could include performing enzyme inhibition or induction studies to confirm the involvement of the identified enzymes. Additionally, genetic studies could be performed to see if individuals with different variants of the enzymes show different rates of metabolite formation.
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