Microsomal assays MCQs With Answer

Microsomal assays MCQs With Answer

Introduction: Microsomal assays are fundamental laboratory tools in drug metabolism and pharmacokinetics, used extensively in M.Pharm curricula to evaluate phase I metabolic stability, cytochrome P450 (CYP)-mediated clearance, and enzyme inhibition. This quiz collection focuses on practical and theoretical aspects of microsomal incubations — including assay design, cofactors, linearity, intrinsic clearance calculations, substrate depletion versus metabolite formation approaches, time-dependent inhibition, and in vitro–in vivo extrapolation (IVIVE). These questions emphasize interpretation of data, common pitfalls, and scaling considerations, helping students sharpen skills needed for experimental planning and critical analysis of microsomal metabolism studies.

Q1. Which component is essential to support cytochrome P450 catalytic activity in human liver microsomal incubations?

• NADPH
• ATP
• UDP-glucuronic acid (UDPGA)
• Glutathione (GSH)

Correct Answer: NADPH

Q2. When performing a microsomal substrate depletion assay to estimate intrinsic clearance, which of the following best describes a primary assumption?

• Metabolite formation is faster than substrate depletion
• Substrate disappearance follows first-order kinetics under assay conditions
• The microsomal protein concentration must be greater than 5 mg/mL
• NADPH must be absent to measure phase II reactions

Correct Answer: Substrate disappearance follows first-order kinetics under assay conditions

Q3. Which of the following is the correct reason to use an NADPH-regenerating system rather than a single bolus of NADPH in microsomal assays?

• Regenerating systems specifically inhibit non-CYP enzymes
• They maintain a steady NADPH concentration, extending linear metabolism period
• They are required for UDP-glucuronosyltransferase activity
• They increase microsomal protein solubility

Correct Answer: They maintain a steady NADPH concentration, extending linear metabolism period

Q4. Which microsomal parameter is commonly used to convert in vitro clearance (CLint, in vitro) to whole liver clearance during IVIVE?

• Plasma protein binding (fup) only
• Microsomal protein per gram liver (MPPGL) combined with liver weight
• Glomerular filtration rate
• Fraction unbound in microsomes (fu,mic) only

Correct Answer: Microsomal protein per gram liver (MPPGL) combined with liver weight

Q5. In a microsomal stability assay, which experimental condition most commonly leads to nonspecific binding of lipophilic compounds and underestimation of intrinsic clearance?

• Low microsomal protein concentration (e.g., 0.05 mg/mL)
• High DMSO percentage (e.g., >1–2% v/v)
• High microsomal protein concentration without correction for binding
• Using phosphate buffer instead of Tris buffer

Correct Answer: High microsomal protein concentration without correction for binding

Q6. Time-dependent inhibition (TDI) of a CYP enzyme in microsomes is most reliably detected by which experimental design?

• Co-incubation of inhibitor and probe substrate with NADPH only at time zero
• Pre-incubation of inhibitor with microsomes and NADPH followed by dilution into probe substrate
• Incubation without NADPH to observe reversible binding
• Pre-incubation of inhibitor with buffer but without microsomes

Correct Answer: Pre-incubation of inhibitor with microsomes and NADPH followed by dilution into probe substrate

Q7. Which parameter derived from a microsomal Michaelis–Menten study best indicates enzyme affinity for a substrate?

• Vmax
• Km
• Intrinsic clearance (Vmax/Km) only
• t1/2 of substrate disappearance

Correct Answer: Km

Q8. For reliable linear kinetics in microsomal incubations, which of the following guidelines is most appropriate?

• Ensure that percent substrate depletion per minute is constant across protein concentrations
• Use the highest possible microsomal protein to maximize signal
• Run reactions until >90% substrate is metabolized
• Always use 37°C for all microsomal assays despite temperature-sensitive substrates

Correct Answer: Ensure that percent substrate depletion per minute is constant across protein concentrations

Q9. Which of the following best describes intrinsic clearance (CLint) estimated from microsomal substrate depletion?

• A measure of clearance limited only by plasma protein binding
• The innate ability of microsomal enzymes to metabolize drug per unit protein under assay conditions
• The observed hepatic clearance in humans without scaling
• A parameter that includes renal excretion contributions

Correct Answer: The innate ability of microsomal enzymes to metabolize drug per unit protein under assay conditions

Q10. Which co-factor is specifically required for UDP-glucuronosyltransferase (UGT) activity in microsomal assays?

• NADH
• NADPH
• UDP-glucuronic acid (UDPGA)
• Phosphoenolpyruvate

Correct Answer: UDP-glucuronic acid (UDPGA)

Q11. During estimation of IC50 for a CYP inhibitor in microsomes, which practice helps to ensure accurate estimation of potency?

• Using a substrate concentration >> Km to reduce sensitivity to competitive inhibitors
• Running assay with no NADPH to prevent metabolism
• Using substrate concentration close to or below Km to reveal competitive inhibition
• Setting microsomal protein extremely high to speed up reaction

Correct Answer: Using substrate concentration close to or below Km to reveal competitive inhibition

Q12. Which statement about pooled human liver microsomes (HLM) is correct?

• Pooled HLM eliminate interindividual variability and reflect an averaged enzyme activity profile
• Pooled HLM only contain CYP3A enzymes and lack UGTs
• Pooled HLM cannot be used for inhibition studies
• Pooled HLM are identical to recombinant single CYP isoform systems

Correct Answer: Pooled HLM eliminate interindividual variability and reflect an averaged enzyme activity profile

Q13. Which mathematical relationship is used to calculate microsomal intrinsic clearance (CLint, mL/min/mg protein) from substrate depletion half-life in a well-mixed incubation?

• CLint = (ln2) / t1/2
• CLint = (ln2 / t1/2) × (incubation volume / microsomal protein mass)
• CLint = Vmax × Km
• CLint = t1/2 × microsomal protein concentration

Correct Answer: CLint = (ln2 / t1/2) × (incubation volume / microsomal protein mass)

Q14. A mechanism-based inactivator typically shows which distinguishing feature in microsomal assays?

• Reversible inhibition that is immediately lost after dilution
• Time-dependent loss of enzyme activity requiring NADPH and showing increased inhibition with pre-incubation time
• Only inhibits non-CYP enzymes
• Does not require cofactors and shows no time dependence

Correct Answer: Time-dependent loss of enzyme activity requiring NADPH and showing increased inhibition with pre-incubation time

Q15. Why is it important to correct microsomal intrinsic clearance for unbound fraction in microsomes (fu,mic) when performing IVIVE for highly lipophilic drugs?

• Because microsomes remove polar metabolites quickly
• Because only unbound drug is available to enzymes; ignoring fu,mic underestimates intrinsic clearance for highly bound drugs
• Because fu,mic corrects for plasma protein binding
• Because fu,mic scales microsomal volume to whole blood volume

Correct Answer: Because only unbound drug is available to enzymes; ignoring fu,mic underestimates intrinsic clearance for highly bound drugs

Q16. Which factor is least likely to affect the outcome of a microsomal stability assay?

• pH of the assay buffer
• Incubation temperature
• Brand of pipette tips used
• Percentage of organic co-solvent (e.g., DMSO)

Correct Answer: Brand of pipette tips used

Q17. In microsomal assays, recombinant CYP enzymes are primarily used to:

• Measure whole-liver clearance directly without scaling
• Identify which specific CYP isoforms metabolize a compound and determine isoform-specific kinetics
• Avoid use of cofactors altogether
• Measure renal clearance pathways

Correct Answer: Identify which specific CYP isoforms metabolize a compound and determine isoform-specific kinetics

Q18. Which experimental observation suggests that observed CYP inhibition is reversible rather than mechanism-based irreversible in microsomes?

• Inhibition increases with pre-incubation time and requires NADPH
• Inhibition is lost after a large dilution into substrate-containing buffer
• Enzyme activity cannot be recovered by dialysis
• Inhibition shows a progressive decrease in Vmax with no change in Km

Correct Answer: Inhibition is lost after a large dilution into substrate-containing buffer

Q19. Which of the following is an appropriate control when measuring UGT-mediated glucuronidation in microsomes?

• Include NADPH-only control
• Include UDPGA-free control to account for non-enzymatic disappearance
• Include glutathione to trap metabolites
• Include high salt to denature microsomes

Correct Answer: Include UDPGA-free control to account for non-enzymatic disappearance

Q20. When scaling in vitro microsomal CLint to predict hepatic clearance using the well-stirred model, which additional parameter is most critical to incorporate?

• Glomerular filtration rate
• Fraction unbound in plasma (fup)
• Microsomal pH only
• Urine flow rate

Correct Answer: Fraction unbound in plasma (fup)

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