Rat liver microsomes (RLM) in metabolite ID MCQs With Answer

Introduction
Rat liver microsomes (RLM) are a vital in vitro system used in drug metabolism and metabolite identification studies in M.Pharm programs. This blog presents targeted MCQs to deepen understanding of how RLM are prepared, how microsomal enzymes (especially cytochrome P450s and ER-associated phase II enzymes) function, and the experimental variables needed for reliable metabolite profiling. Questions cover cofactor requirements, assay setup, common pitfalls (non-specific binding, species differences), analytical strategies for metabolite identification (LC-HRMS), and interpretation for IVIVE and enzyme phenotyping. These MCQs aim to reinforce practical knowledge and analytical reasoning needed for designing and interpreting RLM-based metabolite ID experiments.

Q1. Which class of enzymes is predominantly responsible for drug biotransformation in rat liver microsomes (RLM)?

• Soluble cytosolic enzymes such as glutathione S-transferases
• Mitochondrial dehydrogenases
• Endoplasmic reticulum-associated enzymes, primarily cytochrome P450s (CYPs)
• Plasma esterases

Correct Answer: Endoplasmic reticulum-associated enzymes, primarily cytochrome P450s (CYPs)

Q2. Which cofactor is essential for cytochrome P450-mediated oxidation reactions in microsomal incubations?

• NADH alone
• NADPH or an NADPH-regenerating system
• ATP
• FADH2

Correct Answer: NADPH or an NADPH-regenerating system

Q3. During standard microsome preparation, what centrifugal force is typically used to pellet microsomes from liver homogenate?

• 10,000 × g for 10 minutes
• 20,000 × g for 30 minutes
• 100,000 × g (ultracentrifugation) to pellet microsomes
• 2,000 × g for 5 minutes

Correct Answer: 100,000 × g (ultracentrifugation) to pellet microsomes

Q4. Why are rat liver microsomes commonly used in early metabolite identification before rodent toxicology studies?

• They exactly replicate human metabolism
• They are cheaper but unable to produce phase II conjugates
• They enable detection of rat-specific metabolites and major phase I pathways relevant to preclinical safety
• They contain intact cellular compartments for full-organ metabolism

Correct Answer: They enable detection of rat-specific metabolites and major phase I pathways relevant to preclinical safety

Q5. What is a major limitation of RLM when attempting comprehensive metabolite profiling?

• They over-represent cytosolic phase II reactions like sulfation
• They contain active nuclear enzymes that interfere with assays
• They lack some cytosolic enzymes (e.g., many sulfotransferases), potentially missing certain conjugates
• They cannot perform oxidative metabolism

Correct Answer: They lack some cytosolic enzymes (e.g., many sulfotransferases), potentially missing certain conjugates

Q6. What is the typical orientation of microsomal vesicles after preparation that affects substrate accessibility to CYPs?

• Membrane inverted with luminal face exposed, blocking access to CYP active sites
• Random orientation with half accessible and half inaccessible
• Cytosolic face is predominantly exposed to the incubation medium, allowing CYP access
• Mitochondrial orientation predominates, interfering with ER enzymes

Correct Answer: Cytosolic face is predominantly exposed to the incubation medium, allowing CYP access

Q7. Which cofactor must be added to microsomal incubations to support UDP-glucuronosyltransferase (UGT)-mediated glucuronidation?

• S-adenosylmethionine (SAM)
• Uridine 5′-diphospho-glucuronic acid (UDPGA)
• Glutathione
• NAD+

Correct Answer: Uridine 5′-diphospho-glucuronic acid (UDPGA)

Q8. Non-specific binding of a drug to microsomal protein reduces free substrate concentration. How is this commonly corrected when estimating intrinsic clearance?

• Ignore it because it is negligible
• Measure fraction unbound in microsomes (fu,mic) using equilibrium dialysis or ultrafiltration and correct clearance
• Only measure total drug and assume 100% free
• Dilute microsomes to zero protein concentration

Correct Answer: Measure fraction unbound in microsomes (fu,mic) using equilibrium dialysis or ultrafiltration and correct clearance

Q9. Which in vitro approach is commonly used in microsomes to determine intrinsic clearance (Clint) by monitoring the disappearance of parent drug?

• Equilibrium dialysis method
• Substrate depletion (t1/2) method
• Protein binding assay
• Isothermal titration calorimetry

Correct Answer: Substrate depletion (t1/2) method

Q10. To detect time-dependent (mechanism-based) inhibition of CYPs in RLM, what experimental step is critical?

• Adding MgCl2 to all incubations
• Preincubation with inhibitor in the absence of NADPH
• Preincubation with inhibitor and NADPH before adding probe substrate
• Omitting microsomes during preincubation

Correct Answer: Preincubation with inhibitor and NADPH before adding probe substrate

Q11. Which analytical platform is most widely used for structural identification of metabolites generated from RLM incubations?

• UV-Visible spectrophotometry
• Low-resolution LC with refractive index detection
• Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS, e.g., QTOF or Orbitrap) and MS/MS
• NMR without any chromatographic separation

Correct Answer: Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS, e.g., QTOF or Orbitrap) and MS/MS

Q12. When scaling microsomal intrinsic clearance to predict in vivo hepatic clearance (IVIVE), which parameters are required?

• Only clearance measured in microsomes is required
• Microsomal protein per gram liver, liver weight (or liver blood flow), and a hepatic clearance model (e.g., well-stirred)
• Plasma protein binding only
• Urine excretion rates

Correct Answer: Microsomal protein per gram liver, liver weight (or liver blood flow), and a hepatic clearance model (e.g., well-stirred)

Q13. Which statement best explains why rat liver microsomal metabolite profiles may not fully predict human metabolites?

• Rats have identical CYP isoform expression as humans
• Species differences in CYP and UGT isoforms and expression levels can produce distinct metabolites
• Microsomes cannot perform oxidative metabolism in any species
• Human microsomes cannot form glucuronides

Correct Answer: Species differences in CYP and UGT isoforms and expression levels can produce distinct metabolites

Q14. Which gas is required for cytochrome P450-catalyzed monooxygenation reactions in microsomal incubations?

• Carbon dioxide (CO2)
• Molecular oxygen (O2)
• Nitrogen (N2)
• Hydrogen (H2)

Correct Answer: Molecular oxygen (O2)

Q15. Typical microsomal incubation protein concentrations used for metabolic stability and metabolite ID experiments are often in which range?

• 0.001–0.01 mg/mL
• 0.1–1.0 mg/mL
• 10–20 mg/mL
• 50–100 mg/mL

Correct Answer: 0.1–1.0 mg/mL

Q16. Why is alamethicin sometimes added to microsomal glucuronidation assays?

• To fluorescently label glucuronides
• To permeabilize microsomal membranes and improve cofactor (UDPGA) access for some UGT activities
• To inhibit all phase I enzymes selectively
• To chelate divalent cations

Correct Answer: To permeabilize microsomal membranes and improve cofactor (UDPGA) access for some UGT activities

Q17. Which types of phase I metabolic transformations are most commonly observed in RLM incubations?

• Oxidation (e.g., hydroxylation), N-dealkylation, and dealkylation reactions
• Direct sulfation as the main reaction
• Complete conjugation to glutathione without prior oxidation
• Spontaneous hydrolysis only

Correct Answer: Oxidation (e.g., hydroxylation), N-dealkylation, and dealkylation reactions

Q18. To ensure metabolites observed are enzymatic and not artifacts, which control is essential in RLM experiments?

• Include a control with double the NADPH concentration
• Include incubations with heat-inactivated microsomes or without NADPH as negative controls
• Only run the test compound with buffer and no microsomes
• Do not include any controls to speed up analysis

Correct Answer: Include incubations with heat-inactivated microsomes or without NADPH as negative controls

Q19. For enzyme phenotyping in RLM, which approach is commonly used to implicate specific CYP isoforms responsible for a metabolic pathway?

• Use of selective chemical inhibitors and recombinant human or rat CYPs to confirm involvement
• Assume the most abundant enzyme is responsible without testing
• Perform only genomic sequencing of the rat donor
• Rely solely on substrate depletion without inhibitors or recombinant enzymes

Correct Answer: Use of selective chemical inhibitors and recombinant human or rat CYPs to confirm involvement

Q20. When generating metabolites for structural elucidation, which practice improves the chance of detecting minor metabolites in RLM incubations?

• Use very high protein concentration (>20 mg/mL) without adjusting incubation time
• Run time-course incubations, increase substrate load or scale up incubation volume, and use sensitive LC-HRMS methods
• Exclude cofactors to limit major metabolism
• Only analyze samples with UV detection

Correct Answer: Run time-course incubations, increase substrate load or scale up incubation volume, and use sensitive LC-HRMS methods

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