Metabolic response assays MCQs With Answer

Introduction

This collection of metabolic response assays MCQs with answers is designed for M.Pharm students studying Bioprocess Engineering and Technology. It focuses on experimental methods used to measure cellular metabolism, including respirometry, mitochondrial function tests, colorimetric and fluorometric viability assays, extracellular flux analysis, and metabolite quantification. Questions emphasize assay principles, instrumentation, sample preparation, controls, data interpretation, and common artifacts. The set aims to deepen conceptual understanding and prepare students for practical laboratory work, research design, and critical evaluation of metabolic data in drug development and bioprocessing contexts.

Q1. Which principle best describes the measurement of cellular respiration using a Clark-type oxygen electrode?

• Detection of oxygen-dependent reduction of tetrazolium salts to formazan
• Polarographic measurement of oxygen reduction current at a cathode
• Fluorometric detection of ATP-dependent chemiluminescence
• Optical measurement of pH changes in the medium

Correct Answer: Polarographic measurement of oxygen reduction current at a cathode

Q2. In a Seahorse extracellular flux assay, which two parameters are primarily measured to assess cellular metabolic phenotype?

• Glucose uptake and lactate production
• Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)
• ATP concentration and NADH fluorescence
• Mitochondrial membrane potential and reactive oxygen species

Correct Answer: Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

Q3. During a mitochondrial stress test, which inhibitor is used to determine proton leak-linked respiration by blocking ATP synthase?

• Rotenone
• FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone)
• Oligomycin
• Antimycin A

Correct Answer: Oligomycin

Q4. The MTT assay measures metabolic activity by which biochemical conversion?

• Oxidation of resazurin to resorufin
• Reduction of MTT tetrazolium to insoluble purple formazan by NAD(P)H-dependent oxidoreductases
• Hydrolysis of ATP to adenosine by luciferase
• Production of nitrite from nitrate by bacterial nitrate reductase

Correct Answer: Reduction of MTT tetrazolium to insoluble purple formazan by NAD(P)H-dependent oxidoreductases

Q5. Which limitation is most associated with using tetrazolium-based assays (e.g., MTT, XTT) for assessing cell viability?

• They directly measure mitochondrial ATP concentration which varies independently of viability
• Formazan product solubility and nonuniform reduction rates can lead to assay variability and interference with metabolic modulators
• They require radioactive tracers that complicate disposal
• They exclusively detect extracellular enzymes and miss intracellular metabolism

Correct Answer: Formazan product solubility and nonuniform reduction rates can lead to assay variability and interference with metabolic modulators

Q6. A decrease in basal OCR after treatment with a drug, with unchanged ECAR, most likely indicates which primary effect?

• Enhanced glycolytic flux compensating for mitochondrial inhibition
• Mitochondrial respiratory chain inhibition reducing oxidative phosphorylation
• Increased proton leak across the plasma membrane
• Activation of lactate dehydrogenase increasing lactate export

Correct Answer: Mitochondrial respiratory chain inhibition reducing oxidative phosphorylation

Q7. Which fluorescent probe is commonly used to detect intracellular reactive oxygen species (ROS) and can be oxidized to a fluorescent product?

• JC-1
• DCFDA (2′,7′-dichlorodihydrofluorescein diacetate)
• Rhodamine 123
• Hoechst 33342

Correct Answer: DCFDA (2′,7′-dichlorodihydrofluorescein diacetate)

Q8. When using ATP-luciferase assays for cell viability, which factor is crucial to avoid false-high signals?

• Ensuring sufficient dissolved oxygen in the assay buffer
• Preventing extracellular ATP contamination from lysed cells and serum components and rapidly quenching ATPases
• Maintaining a low pH to stabilize luciferase
• Adding tetrazolium salts to amplify signal

Correct Answer: Preventing extracellular ATP contamination from lysed cells and serum components and rapidly quenching ATPases

Q9. In extracellular flux analysis, what does maximal respiration after FCCP addition represent?

• Non-mitochondrial oxygen consumption
• ATP-linked respiration only
• Maximum electron transport chain capacity when proton gradient is dissipated
• Proton leak-dependent respiration under inhibited ATP synthase

Correct Answer: Maximum electron transport chain capacity when proton gradient is dissipated

Q10. Which assay is most appropriate for quantifying lactate production as an indicator of glycolytic flux in cell culture medium?

• Enzymatic lactate oxidase or lactate dehydrogenase coupled spectrophotometric assay
• JC-1 mitochondrial membrane potential assay
• MTT tetrazolium reduction assay
• DCFDA ROS assay

Correct Answer: Enzymatic lactate oxidase or lactate dehydrogenase coupled spectrophotometric assay

Q11. Which control is essential when interpreting MTT or resazurin reduction assays to distinguish metabolic inhibition from loss of cell number?

• Including a dye-only blank without cells
• Normalizing metabolic readout to total protein or DNA content to account for cell number changes
• Measuring pH of the medium before and after assay
• Using different tetrazolium salts in parallel

Correct Answer: Normalizing metabolic readout to total protein or DNA content to account for cell number changes

Q12. Which statement about mitochondrial membrane potential assays using JC-1 is TRUE?

• JC-1 monomers emit red fluorescence while aggregates emit green fluorescence
• JC-1 aggregates form in depolarized mitochondria producing increased red fluorescence
• JC-1 forms potential-dependent aggregates in polarized mitochondria, shifting emission from green to red
• JC-1 measures ATP directly through luminescence

Correct Answer: JC-1 forms potential-dependent aggregates in polarized mitochondria, shifting emission from green to red

Q13. Which artifact is commonly encountered with fluorescent metabolic probes and can lead to misinterpretation?

• Probe fluorescence is completely independent of probe concentration
• Photobleaching, probe efflux, and nonspecific binding to proteins or plastic can alter signal
• Fluorescent probes always specifically label only the intended metabolite
• All probes are resistant to cellular esterases

Correct Answer: Photobleaching, probe efflux, and nonspecific binding to proteins or plastic can alter signal

Q14. In respirometry experiments, what does the term “non-mitochondrial respiration” refer to?

• Oxygen consumption attributable to processes other than mitochondrial electron transport, such as oxidases and peroxidases
• Total oxygen consumption measured at baseline
• Respiration linked exclusively to complex IV activity
• Proton leak across the inner mitochondrial membrane

Correct Answer: Oxygen consumption attributable to processes other than mitochondrial electron transport, such as oxidases and peroxidases

Q15. Which metabolic assay is most suitable for high-throughput screening of mitochondrial toxicants in a 96-well plate format?

• High-resolution polarographic chamber respirometry for single samples
• Seahorse XF platform for medium-throughput extracellular flux analysis or plate-based ATP assays using luciferase
• Electron microscopy assessment of cristae density
• Radioisotope-based oxygen consumption with 14C-O2

Correct Answer: Seahorse XF platform for medium-throughput extracellular flux analysis or plate-based ATP assays using luciferase

Q16. When performing isotope-tracing metabolomics to study flux, which tracer is commonly used to follow central carbon metabolism?

• 14C-labeled fatty acids only
• 13C-glucose to trace glycolysis, TCA cycle, and related pathways
• 35S-methionine for flux through glycolysis
• Deuterated water exclusively for monitoring electron transport

Correct Answer: 13C-glucose to trace glycolysis, TCA cycle, and related pathways

Q17. LDH release assays are used to assess cell membrane integrity. Which interpretation is most accurate?

• Elevated extracellular LDH indicates increased mitochondrial ATP production
• Increased LDH in supernatant reflects cell membrane damage or necrosis leading to cytosolic enzyme leakage
• LDH release specifically indicates apoptosis without necrosis
• LDH assays quantify mitochondrial respiratory chain activity directly

Correct Answer: Increased LDH in supernatant reflects cell membrane damage or necrosis leading to cytosolic enzyme leakage

Q18. Which calculation is necessary to derive spare respiratory capacity from extracellular flux data?

• Basal OCR minus non-mitochondrial respiration
• Maximal respiration (after FCCP) minus basal respiration
• ECAR at baseline divided by OCR at baseline
• ATP-linked OCR divided by proton leak OCR

Correct Answer: Maximal respiration (after FCCP) minus basal respiration

Q19. Which reagent combination is typically used to completely inhibit mitochondrial respiration in an extracellular flux experiment to measure non-mitochondrial OCR?

• Oligomycin alone
• Rotenone plus antimycin A
• FCCP plus oligomycin
• JC-1 and DCFDA together

Correct Answer: Rotenone plus antimycin A

Q20. When interpreting metabolic assay results across different cell types, which normalization strategy provides the most reliable comparison of per-cell metabolic rates?

• Normalizing to well volume only
• Normalizing to total cell number, or better, to total protein or DNA content per well to account for cell size and number differences
• Using raw fluorescence or absorbance values without normalization
• Normalizing to incubation time only

Correct Answer: Normalizing to total cell number, or better, to total protein or DNA content per well to account for cell size and number differences

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