BCKA Panel Service - Creative Proteomics

Q: What is the difference between BCAAs and BCKAs in measurement strategy?

BCAAs are amino acids quantified without derivatization or with amine-targeting reagents; BCKAs are carbonyl acids that benefit from carbonyl/carboxylate-specific derivatization (e.g., 3-NPH or oxime formation) to improve sensitivity and selectivity in MRM.

Q: Do I need isotope-labeled standards for every analyte?

Isotopologues are preferred for each target. When unavailable, class-specific surrogates can be used with matrix-matched calibration and bias monitoring through spike-recovery experiments.

Q: Can BCKA profiling be integrated with other omics approaches?

Absolutely. Our BCKA panel can be combined with amino acid, acylcarnitine, and TCA intermediate profiling to deliver multi-dimensional insights that strengthen metabolomics or systems biology projects.

Q: How is data reproducibility guaranteed?

Every batch is processed with blanks, spikes, and multi-level QC samples. Results are validated with regression audits, stable-isotope dilution, and cross-matrix comparisons to ensure high confidence in the data.

Q: What is the minimum volume required for low-abundance matrices like CSF?

As little as 80–100 µL per replicate is sufficient with isotope dilution and optimized extraction workflows.

Q: How are solid samples like tissues processed?

We homogenize tissues under cold organic solvents to prevent oxidative degradation and to maximize recovery of BCKAs and related metabolites.

Q: Is this service suitable for exploratory research or only for targeted studies?

The BCKA panel is primarily targeted but can be flexibly expanded with optional metabolites, making it suitable for both exploratory and hypothesis-driven projects.

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What Are BCKAs and Why Measure Them?

Branched-chain keto acids (BCKAs) are the first oxidative deamination products of branched-chain amino acids (BCAAs: valine, leucine, isoleucine). Transamination by BCAT yields three principal BCKAs:

α-ketoisocaproate (KIC) from leucine
α-keto-β-methylvalerate (KMV) from isoleucine
α-ketoisovalerate (KIV) from valine

Because BCKAs sit immediately upstream of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, their absolute and relative concentrations are sensitive indicators of BCAA transamination, BCKDH activity, mitochondrial redox status, and peroxisomal/mitochondrial interplay. Accurate BCKA profiling helps you:

Map BCAA catabolic flux and pathway bottlenecks.
Evaluate nutritional interventions and metabolic engineering strategies.
Monitor cell culture performance in bioprocessing where BCAA utilization affects growth and by-products.
Build and validate mechanistic models of energy metabolism using quantitative, matrix-matched data.

What Problems We Solve for Our Clients

Mechanistic Pathway Research

Possible issue: Unclear BCKDH activity or suspected BCKA accumulation.

Our support: Targeted LC–MS/MS with isotope dilution, optional hydroxy-acids/acylcarnitines, and pathway ratios to reveal flux bottlenecks.

Nutrition & Formulation R&D

Possible issue: Ingredient or dosage effects on BCKA balance are uncertain.

Our support: Comparative BCKA profiling with optional BCAA/acylcarnitine context to clarify oxidation shifts.

Cell Culture & Bioprocess Monitoring

Possible issue: BCKA spikes under changing feed, pH, DO, or C/N conditions.

Our support: Time-series BCKA tracking with trend detection to highlight critical process inflection points.

Microbial Fermentation & Metabolic Engineering

Possible issue: Strain modifications show ambiguous flux redirection.

Our support: Broth analysis of BCKAs, hydroxy-acids, and acylcarnitines, providing ratios that differentiate productive from competing pathways.

Animal & Other Research Models

Possible issue: Unclear distribution of BCKA–BCAA balance across matrices.

Our support: Multi-matrix, isotope-corrected methods ensuring coherent interpretation of tissue and fluid profiles.

Food/Ingredient Research & Stability

Possible issue: Variability between batches or under storage conditions.

Our support: BCKA fingerprinting and similarity scoring to track consistency and detect drift.

What We Offer — BCKA Panel Services

Targeted BCKA Quantitation (Core Panel): KIC, KMV, KIV.
Extended Keto-/Hydroxy-acid Panel (Optional): α-hydroxyisocaproate (HICA), β-hydroxy-β-methylbutyrate (HMB), α-hydroxyisovalerate (HIVA), α-hydroxy-β-methylvalerate (HMV).
Context Metabolites (Optional): BCAAs (Leu/Ile/Val), C5 acylcarnitines (isovaleryl-, 2-methylbutyryl-, isobutyryl-carnitine), selected TCA intermediates.
Stable-Isotope Dilution: ¹³C/²H-labeled analogs for each BCKA when available; class-specific surrogates otherwise.

BCKA Panel and Related Analytes (Measured/Optional)

Group	Analyte (Abbrev)	Synonyms		Reporting Range (ng/mL)	Internal Standard	Stability Notes
Core BCKAs	α-Ketoisocaproate (KIC)	4-methyl-2-oxopentanoate		2 – 5,000	¹³C-KIC	Sensitive to oxidation; freeze promptly
	α-Ketoisovalerate (KIV)	3-methyl-2-oxobutyrate		2 – 5,000	¹³C-KIV	Stable at −80 °C; avoid >2 freeze-thaws
	α-Keto-β-methylvalerate (KMV)	3-methyl-2-oxopentanoate		2 – 5,000	¹³C-KMV	Light-sensitive; ship on dry ice
Derived Hydroxy-/Keto-Acids	α-Hydroxyisocaproate (HICA)	2-hydroxy-4-methylpentanoate		5 – 10,000	¹³C-HICA	Stable under frozen storage
	β-Hydroxy-β-methylbutyrate (HMB)	3-hydroxy-3-methylbutyrate		5 – 10,000	¹³C-HMB	Stable; widely studied in sports nutrition
	α-Hydroxyisovalerate (HIVA)	2-hydroxy-3-methylbutyrate		5 – 10,000	Surrogate	Stable at −80 °C
	α-Hydroxy-β-methylvalerate (HMV)	2-hydroxy-3-methylpentanoate	5 – 10,000		Surrogate	Less common; include if KMV studied
	2-Oxoisocaproylglycine	KIC conjugate		10 – 50,000	Surrogate	Conjugated form; useful in excretion studies
	2-Oxoisovaleroylglycine	KIV conjugate		10 – 50,000	Surrogate	Same as above
Context Amino Acids	Leucine (Leu)	—		10 – 100,000	¹³C-Leu	Stable frozen
	Isoleucine (Ile)	—		10 – 100,000	¹³C-Ile	Stable frozen
	Valine (Val)	—		10 – 100,000	¹³C-Val	Stable frozen
Context Acylcarnitines	Isovalerylcarnitine (C5-IV)	—		1 – 2,000	²H-C5	Stable if stored −80 °C
	2-Methylbutyrylcarnitine (C5-MB)	—		1 – 2,000	²H-C5	Stable frozen
	Isobutyrylcarnitine (C4-IB)	—		1 – 2,000	²H-C4	Stable frozen
	Propionylcarnitine (C3)	—		1 – 2,000	²H-C3	Stable frozen
	Butyrylcarnitine (C4)	—		1 – 2,000	²H-C4	Stable frozen
Related Organic Acids	Succinyl-CoA (proxy via succinate)	—		Variable	Surrogate	Proxy for downstream entry to TCA
	Methylmalonic acid (MMA)	—		5 – 10,000	²H-MMA	Sensitive; monitor B12-related flux

Why Choose Our BCKA Analysis Service: Key Advantages

Sensitivity: Method LLOQs as low as 2 ng/mL for KIC/KIV/KMV in protein-precipitated plasma using LC–MS/MS with derivatization; ≤5 ng/mL in non-derivatized workflows.
Linearity: Weighted 1/x or 1/x² calibration with R² ≥ 0.995 over 3–4 orders of magnitude, matrix-matched.
Precision & Accuracy: Intra-assay CV ≤10%, inter-assay CV ≤15%; mean bias within ±10% across QC levels.
Recovery & Matrix Effects: Absolute recovery typically 80–110%; ion suppression/ enhancement corrected via stable-isotope dilution and post-extraction spiking.
Carryover Control: Analyte carryover maintained <0.1% of low-cal calibrators with programmed needle wash and diversion valves.
Stability Provenance: Freeze–thaw stability within ±15% across ≥3 cycles; short-term bench stability established for each matrix/derivative chemistry.
Traceable Quantitation: Calibration traceability to NMI-linked reference materials or in-house characterized stocks with documented purity assessment.

Key Instrument Platforms for Accurate BCKA Panel Quantification

Creative Proteomics applies targeted LC–MS/MS as the primary platform, with GC–MS/MS as an orthogonal option for confirmation or special matrices.

LC–MS/MS (Main Approach):
Using UHPLC systems coupled with high-sensitivity triple quadrupoles, α-keto acids are stabilized through derivatization (e.g., 3-NPH), quantified with isotope-labeled internal standards, and measured in scheduled MRM mode to ensure selectivity and reproducibility.
GC–MS/MS (Complementary):
Used for confirmation of certain organic acids or for legacy comparability, applying derivatization to enhance volatility and sensitivity.

Key advantages for clients:

High sensitivity (LLOQ down to ~2 ng/mL for BCKAs).
Robust quantitation with isotope dilution calibration.
Flexibility to adapt workflows across plasma, serum, tissue, cells, fermentation broth, and formulated products.

Waters ACQUITY UPLC System (Figure from Waters)

SCIEX Triple Quad™ 6500+ (Figure from Sciex)

Agilent 1260 Infinity II HPLC (Figure from Agilent)

Agilent 6495C Triple quadrupole (Figure from Agilent)

How Our BCKA Panel Assay Works — Step-by-Step Process

BCKA Panel Analysis Workflow

Sample Requirements for BCKA Panel

Matrix	Preferred Container	Minimum Amount	Pretreatment Notes	Storage & Shipping
Plasma/Serum	Polypropylene cryovial	80–150 µL per replicate	EDTA or heparin; avoid citrate for carboxylate analysis	Freeze ≤ −70 °C; ship on dry ice
CSF	Polypropylene cryovial	80–150 µL	Handle low protein carefully; avoid repeated thaw	Freeze ≤ −70 °C; ship on dry ice
Tissue	Cryovial / tube with beads	≥ 20 mg (wet)	Snap-frozen; we homogenize under cold organic solvent	Keep frozen; ship on dry ice
Cultured Cells	Pellet in cryovial	≥ 1×10^6 cells	Wash with cold PBS; quench and pellet rapidly	Keep frozen; ship on dry ice
Culture Media / Fermentation Broth	Plastic vial	≥ 300 µL	Record formulation; filter particulates if possible	Chill or freeze; ship cold/frozen
Formulated Products / Nutritional Materials	Plastic vial	≥ 200 mg or ≥ 300 µL	Provide ingredient list for interferences check	Protect from light; ship cold/frozen

Deliverables: What You Receive from Our BCKA Analysis

Certificate of Analysis (RUO): Analyte list, method synopsis, calibration model, reportable ranges.
Concentration Tables: Absolute values and optional ratios (e.g., KIC/KIV, ΣBCKA/ΣBCAA) in your requested units.
QC & Method Performance Summary: Linearity, precision, accuracy, recovery, and matrix-effect data for the batch.
Chromatograms & Spectra: Representative MRM traces; retention-time windows and qualifiers.
Raw Data Package: Vendor files and processed exports (mzML, CSV/XLSX) to enable re-analysis.
Study Notes: Any observed interferences, out-of-trend samples, or recommendations for follow-up.

Box plot of reduced (GSH) and oxidized glutathione (GSSG) concentrations in different sample groups.

LOD curves of KIC, KIV, KMV with threshold line and detection points.

Standard calibration curve of GSH with regression line and R² for LC–MS/MS quantification.

Chromatogram with three peaks (KIC, KIV, KMV) and mass spectrum with ions at m/z 86, 102, 144.

Bar and line charts of KIC, KIV, KMV concentrations and ratios (KIC/KIV, ΣBCKA/ΣBCAA) across groups.

QC summary showing accuracy, precision (CV%), and recovery for BCKA assays.

Heatmap of KIC, KIV, KMV levels in plasma, serum, tissue, cells, and fermentation samples.

Grouped bar chart comparing KIC, KIV, KMV concentrations in control and treatments.

What is the difference between BCAAs and BCKAs in measurement strategy?

BCAAs are amino acids quantified without derivatization or with amine-targeting reagents; BCKAs are carbonyl acids that benefit from carbonyl/carboxylate-specific derivatization (e.g., 3-NPH or oxime formation) to improve sensitivity and selectivity in MRM.

Do I need isotope-labeled standards for every analyte?

Isotopologues are preferred for each target. When unavailable, class-specific surrogates can be used with matrix-matched calibration and bias monitoring through spike-recovery experiments.

Can BCKA profiling be integrated with other omics approaches?

Absolutely. Our BCKA panel can be combined with amino acid, acylcarnitine, and TCA intermediate profiling to deliver multi-dimensional insights that strengthen metabolomics or systems biology projects.

How is data reproducibility guaranteed?

Every batch is processed with blanks, spikes, and multi-level QC samples. Results are validated with regression audits, stable-isotope dilution, and cross-matrix comparisons to ensure high confidence in the data.

What is the minimum volume required for low-abundance matrices like CSF?

As little as 80–100 µL per replicate is sufficient with isotope dilution and optimized extraction workflows.

How are solid samples like tissues processed?

We homogenize tissues under cold organic solvents to prevent oxidative degradation and to maximize recovery of BCKAs and related metabolites.

Is this service suitable for exploratory research or only for targeted studies?

The BCKA panel is primarily targeted but can be flexibly expanded with optional metabolites, making it suitable for both exploratory and hypothesis-driven projects.

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