β-Oxidation Flux Panel Service

What is β-Oxidation Flux Analysis and Why It Matters

β-Oxidation flux analysis is a targeted metabolomics approach that quantifies the dynamic rate at which fatty acids are broken down via the β-oxidation pathway in mitochondria and peroxisomes. Instead of measuring only static metabolite concentrations, this method uses stable isotope tracers—such as [U-¹³C]-palmitate or [U-¹³C]-oleate—combined with high-sensitivity LC–MS/MS to track carbon flow through fatty acid activation, transport, and sequential oxidation cycles, ultimately generating acetyl-CoA for the tricarboxylic acid (TCA) cycle and downstream energy production.

In many research contexts, static lipid measurements cannot reveal pathway capacity, directionality, or bottlenecks. β-Oxidation flux analysis addresses this by providing quantitative, time-resolved insight into functional FAO activity, mitochondrial versus peroxisomal contributions, and metabolic rerouting under genetic, nutritional, or chemical perturbations. This is essential for understanding energy metabolism, optimizing bioprocess efficiency, validating mechanism-of-action studies, and guiding metabolic engineering strategies.

What Problems We Solve for Our Clients

Quantify Fatty Acid Oxidation Activity

Determine FAO pathway throughput under genetic perturbations, nutrient shifts, or compound exposure.

Typical readouts: fractional enrichment of acetyl-CoA surrogates, citrate M+2 propagation, condition-to-control fold changes, acylcarnitine pattern shifts.

Differentiate Mitochondrial and Peroxisomal Contributions

Resolve pathway participation using chain-length–specific acylcarnitines and dicarboxylic acids.

Typical readouts: VLCFA acylcarnitine signatures, ω-oxidation markers, relative contribution estimates derived from isotopologue and analyte patterns.

Trace Carbon Flow into Central Metabolism

Map how [U-¹³C] fatty acids label acetyl-CoA and propagate into the TCA cycle and ketogenesis.

Typical readouts: isotopologue distributions (MIDs) for citrate, malate, succinate; ketone body labeling where applicable.

Locate Rate-Limiting Steps and Metabolic Bottlenecks

Identify constraints across CPT1/2 transport and acyl-CoA dehydrogenase nodes (VLCAD/LCAD/MCAD/SCAD).

Typical readouts: accumulation of chain-matched acylcarnitines, intermediate-to-product ratios, flux sensitivity analysis.

Validate On-Target Pharmacology for FAO-Modulating Candidates

Confirm mechanism engagement without clinical claims.

Typical readouts: restoration or enhancement of pathway labeling, normalization of bottleneck-associated acylcarnitines.

Optimize Process Energy Balance in Cell Systems

Compare media or feed strategies for metabolic efficiency.

Typical readouts: FAO-linked enrichment vs. viability/production KPIs, acylcarnitine burden reduction.

Service Scope — Creative Proteomics β-Oxidation Flux Panel

• FAO Flux Quantification – Stable isotope tracing with ¹³C-labeled fatty acids, enrichment metrics, and optional model-based flux estimation.
• Acylcarnitine Profiling – Chain-length–resolved targeted profiling via LC–MS/MS with internal standard calibration.
• CoA Ester Quantitation – Quantification of key CoA esters using targeted MS methods with high accuracy and precision.
• TCA Isotopologue Mapping – Tracking carbon label incorporation into TCA intermediates to assess downstream metabolic flux.
• Mitochondrial vs Peroxisomal Contribution Assessment – Determine relative pathway activity using targeted acylcarnitine and dicarboxylic acid profiles. Tracer selection (e.g., chain length) can be tailored to emphasize mitochondrial or peroxisomal β-oxidation.
• Ketogenesis Readout – Measuring ketone body production and labeling to evaluate acetyl-CoA overflow.
• Multi-Tracer Substrate Competition Designs – Combining multiple labeled substrates to investigate fuel utilization and pathway interactions.
• Peroxisomal β-Oxidation Module – Extended coverage for very-long-chain fatty acid oxidation activity.

β-Oxidation Flux Panel – Analyte Coverage

Analyte classes covered in each module are detailed below:

Tracer Options and Study Modes

Primary ¹³C Tracers: [U-¹³C]-palmitate, [U-¹³C]-oleate, [1-¹³C]-palmitate.

Deuterated Standards: e.g., d₃-carnitine and chain-matched labeled acylcarnitines for accurate quantitation.

Static plus Flux Hybrid: Combine unlabeled quantitation with stable-isotope tracing for capacity and utilization in one design.

Multi-Tracer Designs: Use multiple labeled substrates to study fuel preference and metabolic rerouting.

Why Choose Our β-Oxidation Flux Panel: Key Advantages

• High Sensitivity Detection – Detects low nanomolar levels of acylcarnitines and organic acids.
• Excellent Quantitative Accuracy – Strong linearity with isotopically labeled internal standards.
• Reproducible Results – Consistent performance across batches.
• Comprehensive Analyte Coverage – Over 40 targeted species across short-, medium-, long-, and very-long-chain acylcarnitines, plus CoA esters, ketone bodies, and TCA intermediates.
• Dual-Platform Confidence – UHPLC–MRM quantitation plus optional HRMS confirmation.
• Isotopologue Precision – Reliable MID measurement with natural-abundance correction.
• Flexible Tracer Options – Multiple ¹³C-labeled fatty acids and multi-tracer designs.

Technical Specifications You Can Rely On

Platforms & Acquisition

• UHPLC–MS/MS on triple quadrupole (MRM) for targeted quantitation, with high-resolution MS (Orbitrap/Q-TOF) for confirmatory ID when requested.
• Dual-polarity switching with retention-time scheduling for maximal panel coverage.
• Calibration: Multi-point external calibration with isotopically labeled internal standards; typical linearity R² ≥ 0.995.
• Sensitivity: Method LOD/LOQ in the low nM to sub-µM range for acylcarnitines and many organic acids (matrix-dependent).
• Precision: Intra-/inter-batch %CV targets ≤ 15% for the majority of analytes in qualified matrices.
• Carryover Control: Assessed each batch with blanks and post-run rinses; acceptance criteria reported.

Flux Analytics

• MID extraction with isotope-correction; enrichment metrics presented with propagated error.
• Optional model-based flux estimation using established MFA tools (e.g., INCA/IsoCor-compatible outputs), with sensitivity analyses.

SCIEX Triple Quad™ 6500+ (Figure from Sciex)

Agilent 6495C Triple quadrupole (Figure from Agilent)

Waters ACQUITY UPLC System (Figure from Waters)

Agilent 1260 Infinity II HPLC (Figure from Agilent)

How Our β-Oxidation Flux Analysis Works — Step-by-Step Process

How to Prepare and Ship Samples for β-Oxidation Flux Studies

Note:

• Labeling Information: Provide tracer type, concentration, and labeling duration with the submission form.
• General Handling: Avoid repeated freeze–thaw cycles. Minimize processing time before freezing. Always ship on dry ice in secondary containment to prevent leakage.

Applications of β-Oxidation Flux Panel

Mechanistic Metabolism Research

Quantify fatty acid oxidation rates and pathway engagement under genetic modifications, nutrient changes, or compound exposure.

Mitochondrial vs Peroxisomal Function Studies

Distinguish contributions of different organelles to β-oxidation and downstream carbon flow.

Metabolic Flux Mapping

Trace ¹³C-labeled fatty acids into TCA cycle intermediates and ketone bodies to understand carbon redistribution.

Pharmacology and Mode-of-Action Validation

Confirm target engagement for FAO-modulating compounds through isotopologue patterns and acylcarnitine profiles.

Bioprocess and Cell Culture Optimization

Monitor FAO activity to optimize media composition, energy balance, and productivity in cell systems.

Metabolic Engineering

Evaluate engineered strains or cell lines for enhanced lipid utilization, energy efficiency, or alternative pathway engagement.

Deliverables: What You Get from Our β-Oxidation Flux Analysis Service

β-oxidation, TCA cycle, and ketogenesis pathway map with enriched metabolites highlighted.

Stacked isotopologue distributions (M+0, M+2, M+4) for citrate, malate, succinate, α-ketoglutarate, and β-hydroxybutyrate.