Human liver microsomes (HLM) in metabolite ID MCQs With Answer

Introduction

Human liver microsomes (HLM) are a cornerstone in preclinical drug metabolism studies, especially for metabolite identification (MetID). This quiz set focuses on practical and theoretical aspects of using HLM for Phase I/II biotransformation, common incubation strategies, cofactor requirements, enzyme phenotyping, and pitfalls such as non-specific binding, reactive metabolite trapping, and scale-up for IVIVE. Designed for M.Pharm students studying Modern Bio‑Analytical Techniques, these MCQs emphasize mechanistic understanding and experimental design—helpful for lab planning, interpreting LC‑MS metabolite profiles, and linking in vitro findings to in vivo metabolism. The questions aim to deepen comprehension beyond rote facts to applied problem solving.

Q1. What is the most common centrifugal force used to prepare microsomes from liver homogenate?

• 10,000 g
• 100,000 g
• 1,000 g
• 50,000 g

Correct Answer: 100,000 g

Q2. Which cofactor is essential for cytochrome P450 (CYP)-mediated reactions in HLM incubations?

• UDP‑glucuronic acid (UDPGA)
• NADPH
• PAPS (3′-phosphoadenosine‑5′‑phosphosulfate)
• Acetyl‑CoA

Correct Answer: NADPH

Q3. For glucuronidation studies with HLM, which addition commonly improves access of UDPGA to luminal UGT active sites?

• β‑mercaptoethanol
• Alamethicin
• Glutathione
• EDTA

Correct Answer: Alamethicin

Q4. Which phase II enzyme is predominantly cytosolic and therefore not well represented in microsomal preparations?

• UDP‑glucuronosyltransferase (UGT)
• Sulfotransferase (SULT)
• Microsomal epoxide hydrolase (mEH)
• CYP3A4

Correct Answer: Sulfotransferase (SULT)

Q5. In substrate depletion studies with HLM, which parameter is directly used to estimate intrinsic clearance (Clint) from the depletion rate?

• t1/2 (half‑life) or apparent first‑order elimination rate constant
• Maximum velocity (Vmax)
• Michaelis constant (Km)
• Partition coefficient (LogP)

Correct Answer: t1/2 (half‑life) or apparent first‑order elimination rate constant

Q6. When scaling microsomal Clint to whole liver intrinsic clearance for IVIVE, which hepatic scaling factor is commonly used (approximate human value)?

• 0.5 mg microsomal protein per gram liver
• 45 mg microsomal protein per gram liver
• 450 mg microsomal protein per kg body weight
• 0.045 mg microsomal protein per gram liver

Correct Answer: 45 mg microsomal protein per gram liver

Q7. Which experimental control is most important to confirm that a metabolite formation in HLM is enzyme‑dependent?

• Heat‑inactivated microsomes control
• PBS only control
• Extra NADPH added control
• Half concentration of substrate control

Correct Answer: Heat‑inactivated microsomes control

Q8. Which approach is best for identifying reactive electrophilic metabolites formed in HLM incubations?

• Include glutathione (GSH) as a trapping agent and search for GSH adducts by LC‑MS
• Omit cofactors to prevent metabolism
• Use only recombinant UGT enzymes
• Perform incubations at 4 °C to stabilize intermediates

Correct Answer: Include glutathione (GSH) as a trapping agent and search for GSH adducts by LC‑MS

Q9. Pooled HLM from multiple donors are used primarily to:

• Increase activity of a single polymorphic enzyme
• Reduce individual donor variability and provide average human metabolism
• Isolate a specific CYP isoform
• Eliminate the need for cofactors in incubations

Correct Answer: Reduce individual donor variability and provide average human metabolism

Q10. Which of the following is a major limitation of HLM for full metabolite profiling?

• Lack of phase I enzymes
• Poor representation of cytosolic enzymes like NAT and many SULTs
• Inability to support CYP reactions
• Incompatibility with LC‑MS detection

Correct Answer: Poor representation of cytosolic enzymes like NAT and many SULTs

Q11. Which experimental parameter most directly helps maintain linear metabolite formation during an HLM incubation?

• Using high substrate concentration near solubility limit
• Short incubation time and low microsomal protein to stay within linear initial rate conditions
• Incubating at room temperature for longer periods
• Adding detergents to increase protein concentration

Correct Answer: Short incubation time and low microsomal protein to stay within linear initial rate conditions

Q12. For enzyme phenotyping of a CYP‑mediated pathway observed in HLM, which is the clearest approach?

• Use chemical inhibitors selective for candidate CYPs and/or recombinant human CYPs to confirm activity
• Vary ionic strength of the buffer
• Always assume CYP3A4 is responsible because it is abundant
• Use only pooled HLM without any specific inhibitors

Correct Answer: Use chemical inhibitors selective for candidate CYPs and/or recombinant human CYPs to confirm activity

Q13. Which statement about non‑specific binding of lipophilic drugs to microsomes is correct?

• Non‑specific binding is negligible and can be ignored for lipophilic drugs
• Non‑specific binding can reduce unbound substrate concentration and should be corrected when estimating intrinsic clearance
• Non‑specific binding increases apparent Vmax
• Non‑specific binding only occurs with hydrophilic drugs

Correct Answer: Non‑specific binding can reduce unbound substrate concentration and should be corrected when estimating intrinsic clearance

Q14. Which analytical technique is the most suitable for structural elucidation of metabolites generated in HLM incubations?

• UV spectroscopy alone
• LC‑HRMS/MS (liquid chromatography–high resolution tandem mass spectrometry)
• Fluorimetry without separation
• Gel electrophoresis

Correct Answer: LC‑HRMS/MS (liquid chromatography–high resolution tandem mass spectrometry)

Q15. Which of the following additions is required to study sulfation reactions using liver preparations?

• UDPGA
• PAPS
• NADPH‑regenerating system
• GSH

Correct Answer: PAPS

Q16. If a compound shows formation of multiple metabolites in HLM but none in recombinant CYP assays, what is a plausible explanation?

• Metabolism is non‑enzymatic
• Multiple enzyme classes (e.g., UGTs, mEH, or flavin monooxygenases) present in microsomes but not in that specific recombinant CYP panel are responsible
• The HLM were contaminated with renal microsomes
• The compound is unstable in organic solvent only

Correct Answer: Multiple enzyme classes (e.g., UGTs, mEH, or flavin monooxygenases) present in microsomes but not in that specific recombinant CYP panel are responsible

Q17. Which practice improves detection of low‑abundance metabolites in HLM incubations analyzed by LC‑MS?

• Use large injection volumes without sample cleanup
• Apply sample concentration/enrichment (e.g., SPE) and use high‑resolution MS with data‑dependent acquisition
• Reduce chromatographic separation time to under 1 minute
• Eliminate cofactors to prevent background ions

Correct Answer: Apply sample concentration/enrichment (e.g., SPE) and use high‑resolution MS with data‑dependent acquisition

Q18. When comparing HLM lots from different donors, which factor most commonly causes variability in metabolic rates?

• Buffer pH, which is always different between lots
• Differences in expression levels of specific drug‑metabolizing enzymes such as polymorphic CYPs
• Different supplier packaging colors
• Microsomes universally have identical enzyme content across donors

Correct Answer: Differences in expression levels of specific drug‑metabolizing enzymes such as polymorphic CYPs

Q19. Which statement about using S9 fraction versus HLM for metabolite ID is correct?

• HLM contains both cytosolic and microsomal enzymes, whereas S9 only contains microsomal enzymes
• S9 fraction includes both cytosolic and microsomal enzymes and may detect phase II metabolites not seen in HLM
• S9 cannot support glucuronidation
• S9 is purified microsomes and therefore identical to HLM

Correct Answer: S9 fraction includes both cytosolic and microsomal enzymes and may detect phase II metabolites not seen in HLM

Q20. Which factor would you adjust if you observe substrate depletion is too fast in HLM and metabolites are below detection limits?

• Increase microsomal protein concentration
• Decrease microsomal protein concentration or shorten incubation time to slow depletion and capture metabolites
• Raise incubation temperature to accelerate formation
• Add excess NADPH to drive complete metabolism

Correct Answer: Decrease microsomal protein concentration or shorten incubation time to slow depletion and capture metabolites

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