Assay of drug-metabolizing enzymes MCQs With Answer

Introduction: Assay of drug-metabolizing enzymes MCQs With Answer is designed for M.Pharm students preparing for Modern Bio-Analytical Techniques (MPA 202T). This set of questions covers in vitro and in vivo assay principles for phase I and phase II enzymes, common probe substrates, cofactors, assay formats (microsomes, S9, hepatocytes, recombinant enzymes), analytical detection methods (LC-MS/MS, radiometric, fluorescence, luminescence), kinetic parameters (Km, Vmax, CLint), and practical considerations such as linearity, inhibition, and induction assays. The MCQs are crafted to deepen conceptual understanding and practical troubleshooting skills needed for designing and interpreting enzyme assay data in drug metabolism studies.

Q1. Which analytical method is most commonly used to quantify metabolites formed in in vitro cytochrome P450 assays with high sensitivity and specificity?

• LC-MS/MS
• UV-Visible spectrophotometry
• Nuclear magnetic resonance (NMR)
• ELISA

Correct Answer: LC-MS/MS

Q2. Which probe substrate is most frequently used to assess CYP3A4 catalytic activity in vitro?

• Midazolam
• Dextromethorphan
• Chlorzoxazone
• Phenacetin

Correct Answer: Midazolam

Q3. Which cofactor must be added to in vitro UDP-glucuronosyltransferase (UGT) assays to enable glucuronidation?

• UDP-glucuronic acid (UDPGA)
• 3′-Phosphoadenosine-5′-phosphosulfate (PAPS)
• NADPH
• Reduced glutathione (GSH)

Correct Answer: UDP-glucuronic acid (UDPGA)

Q4. What does the term “S9 fraction” refer to in drug metabolism studies?

• Post-mitochondrial supernatant from tissue homogenate containing both cytosol and microsomes
• Isolated microsomal pellet rich in CYP enzymes
• Purified mitochondrial fraction
• Plasma fraction free of cellular components

Correct Answer: Post-mitochondrial supernatant from tissue homogenate containing both cytosol and microsomes

Q5. In enzyme kinetics, which parameter corresponds to the substrate concentration at which the reaction velocity is half of Vmax?

• Km
• Vmax
• kcat
• CLint

Correct Answer: Km

Q6. Time-dependent inhibition (TDI) of CYP enzymes is typically detected by which experimental approach?

• Preincubation of inhibitor with enzyme in presence of NADPH before adding substrate
• Single-point IC50 measurement without preincubation
• Measuring mRNA levels after 24 hours
• Using PAPS instead of NADPH

Correct Answer: Preincubation of inhibitor with enzyme in presence of NADPH before adding substrate

Q7. Which probe reaction is commonly used to measure CYP1A2 activity?

• Phenacetin O-deethylation
• Midazolam 1′-hydroxylation
• Dextromethorphan O-demethylation
• Chlorzoxazone 6-hydroxylation

Correct Answer: Phenacetin O-deethylation

Q8. Which assay format uses luminescent substrates engineered to produce light upon CYP-catalyzed metabolism?

• P450-Glo luminescence assay
• EROD fluorescence assay
• Radiometric 14C substrate assay
• HPLC-UV absorbance assay

Correct Answer: P450-Glo luminescence assay

Q9. Which phase II enzyme catalyzes conjugation of electrophilic compounds with reduced glutathione?

• Glutathione S-transferase (GST)
• Sulfotransferase (SULT)
• UDP-glucuronosyltransferase (UGT)
• N-acetyltransferase (NAT)

Correct Answer: Glutathione S-transferase (GST)

Q10. Which in vitro assay is commonly used to estimate hepatic intrinsic clearance of a drug via metabolic stability?

• Microsomal stability assay
• Caco-2 permeability assay
• Plasma protein binding assay
• Hemolysis assay

Correct Answer: Microsomal stability assay

Q11. Which in vitro system is considered the gold standard for studying enzyme induction relevant to human liver?

• Primary human hepatocytes
• Human liver microsomes
• Recombinant CYP-expressing bacteria
• Rodent hepatoma cell line

Correct Answer: Primary human hepatocytes

Q12. Which cofactor is essential for sulfotransferase (SULT) activity in phase II metabolism assays?

• 3′-Phosphoadenosine-5′-phosphosulfate (PAPS)
• UDP-glucuronic acid (UDPGA)
• NADH
• FMN

Correct Answer: 3′-Phosphoadenosine-5′-phosphosulfate (PAPS)

Q13. What is the most appropriate modern approach to accurately determine the enzyme inhibition constant (Ki)?

• Nonlinear regression modeling of velocity versus inhibitor concentration
• Single-point IC50 estimation only
• Lineweaver–Burk double reciprocal linear plot exclusively
• Qualitative gel electrophoresis

Correct Answer: Nonlinear regression modeling of velocity versus inhibitor concentration

Q14. Radiometric assays often employ which labelled isotope to track parent drug disappearance or metabolite formation?

• Carbon-14 (14C)
• Phosphorus-32 (32P)
• Uranium-238 (238U)
• Chlorine-35 (35Cl)

Correct Answer: Carbon-14 (14C)

Q15. Which experimental condition is critical to maintain to ensure initial-rate linearity in microsomal metabolic assays?

• Protein concentration and incubation time within the linear range
• Using extremely high substrate concentrations above solubility limits
• Omitting cofactors such as NADPH
• Incubating at room temperature without mixing

Correct Answer: Protein concentration and incubation time within the linear range

Q16. Recombinant human CYP enzymes are primarily used in metabolism studies to:

• Identify which specific CYP isoform metabolizes a drug
• Measure plasma protein binding
• Assess blood–brain barrier permeability
• Determine renal clearance

Correct Answer: Identify which specific CYP isoform metabolizes a drug

Q17. Which substrate is commonly used in a spectrophotometric assay to measure glutathione S-transferase (GST) activity?

• 1-Chloro-2,4-dinitrobenzene (CDNB)
• Midazolam
• Phenacetin
• UDP-glucuronic acid

Correct Answer: 1-Chloro-2,4-dinitrobenzene (CDNB)

Q18. Which additive is commonly included in microsomal UGT assays to permeabilize the microsomal membrane and allow UDPGA access to active sites?

• Alamethicin
• NADPH
• PAPS
• EDTA

Correct Answer: Alamethicin

Q19. Intrinsic clearance (CLint) calculated from in vitro Michaelis–Menten parameters is best represented by which expression under low substrate concentrations?

• Vmax / Km
• Km / Vmax
• Vmax × Km
• Vmax − Km

Correct Answer: Vmax / Km

Q20. Which direct measurement provides the most functional evidence of cytochrome P450 enzyme induction in hepatocyte cultures?

• Increase in enzyme catalytic activity measured using a specific probe substrate
• Decrease in medium pH
• Increase in cell proliferation rate
• Change in culture color

Correct Answer: Increase in enzyme catalytic activity measured using a specific probe substrate

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