Thiaminase-1 Antibody

What is thiaminase-1 and why are antibodies against it important in research?

Thiaminase-1 is an enzyme that catalyzes the degradation of thiamine (vitamin B1) by cleaving the molecule between its thiazole and pyrimidine moieties. The enzyme plays a crucial role in certain metabolic reactions and has been identified in various microorganisms, including certain bacteria like Bacillus thiaminolyticus and Burkholderia thailandensis .

Antibodies against thiaminase-1 are important research tools that enable detection, quantification, and localization of this enzyme in biological samples. These antibodies facilitate investigations into thiaminase-1's biological functions, expression patterns in different tissues, and potential roles in disease states, particularly in conditions associated with thiamine deficiency or in cancer research where thiaminase-1 has shown potential therapeutic applications .

In which organisms is thiaminase-1 naturally expressed?

Thiaminase-1 is primarily produced by a subset of microorganisms, including certain bacteria like Burkholderia thailandensis and Bacillus thiaminolyticus . Interestingly, while the enzyme is only known to be produced by microorganisms, thiaminase-1 activity has been detected in various plant and animal tissues .

Research suggests that the thiaminase-1 activity observed in plants and animals may actually originate from their microbiota rather than being endogenously produced by the host organisms themselves . This raises important considerations for researchers investigating thiaminase-1 in complex biological systems, as they must account for potential microbial contributions when analyzing enzyme activity in tissue samples.

How does thiaminase-1 differ from other thiamine-degrading enzymes?

Thiaminase-1 specifically cleaves thiamine between its thiazole and pyrimidine moieties, releasing these precursors which can then be salvaged for thiamine synthesis . This mechanism distinguishes it from other thiamine-metabolizing enzymes that may phosphorylate, dephosphorylate, or otherwise modify thiamine without fully cleaving the molecule.

Unlike some other metabolic enzymes, thiaminase-1 demonstrates versatility in its ability to process not only thiamine itself but also phosphorylated forms of thiamine and certain thiamine analogs . This broader substrate range has implications for researchers designing inhibitor studies or investigating the enzyme's biological roles in different contexts.

What are the optimal methods for detecting thiaminase-1 expression in biological samples?

Detection of thiaminase-1 in biological samples can be accomplished through several complementary approaches:

• Enzymatic Activity Assays: Spectrophotometric assays measuring changes in absorbance at 252 nm can assess thiaminase-1 activity by monitoring the reaction of thiamine with secondary nucleophiles . This approach detects functional enzyme activity rather than mere presence.

• Immunological Detection: Using thiaminase-1 specific antibodies, Western blotting can identify and quantify the enzyme in protein extracts. Immunohistochemistry or immunofluorescence techniques enable visualization of thiaminase-1 distribution within tissues or cells.

• mRNA Expression Analysis: RT-PCR or RNA-seq methodologies can detect thiaminase-1 gene expression at the transcriptional level, providing insights into regulation patterns.

For optimal sensitivity and specificity, researchers should consider combining multiple detection methods. When using antibody-based approaches, validation with appropriate positive and negative controls is essential to ensure specificity, particularly given the restricted distribution of thiaminase-1 across species.

How can recombinant thiaminase-1 be efficiently produced and purified for research applications?

Efficient production and purification of recombinant thiaminase-1 involves several key steps, as demonstrated in published research:

• Expression System Selection: E. coli BL21 (DE3) with an IPTG-inducible expression vector (such as pET22b(+)) has been successfully used for thiaminase-1 overexpression . The inclusion of an N-terminal polyhistidine tag facilitates detection and purification.

• Expression Optimization: Following IPTG induction, culture conditions (temperature, duration, media composition) should be optimized to maximize soluble protein expression while minimizing inclusion body formation.

• Purification Protocol:

  • Cell lysis to release the recombinant protein

  • Affinity chromatography using HisTrap FF columns or similar histidine-tag binding matrices

  • Elution with an imidazole gradient

  • Purity assessment via SDS-PAGE

• Activity Verification: The purified enzyme should be tested for activity using spectrophotometric assays based on established methods like those developed by Lienhard and modified by Costello et al.

Maintaining proper storage conditions (-80°C with appropriate cryoprotectants) is essential for preserving enzymatic activity for longer-term research applications.

What controls should be included when using thiaminase-1 antibodies in experimental procedures?

When using thiaminase-1 antibodies in research, the following controls are essential:

• Positive Controls:

  • Purified recombinant thiaminase-1 protein

  • Lysates from organisms known to express thiaminase-1 (e.g., B. thailandensis or B. thiaminolyticus)

  • Tissues or cell lines with confirmed thiaminase-1 expression

• Negative Controls:

  • Samples from thiaminase-1 knockout models

  • Cell lines or tissues known not to express thiaminase-1

  • For bacterial studies, mutant strains with thiaminase-1 gene deletion (like the BT10432 strain mentioned in the research)

• Antibody Validation Controls:

  • Pre-absorption with purified antigen to confirm specificity

  • Secondary antibody-only controls to identify non-specific binding

  • Isotype controls to distinguish specific from non-specific interactions

• Methodological Controls:

  • For activity assays, include heat-inactivated enzyme samples

  • When studying bacterial thiaminase-1, compare wild-type with thiaminase-1 mutant strains as done in B. thailandensis research

These controls help ensure the reliability and specificity of results obtained using thiaminase-1 antibodies, particularly important given the limited distribution of this enzyme across species.

How can thiaminase-1 antibodies be used to investigate cellular responses to thiamine deficiency?

Thiaminase-1 antibodies provide valuable tools for studying cellular responses to thiamine deficiency through several sophisticated approaches:

• Monitoring Compensatory Expression: Antibodies can detect potential upregulation or downregulation of thiaminase-1 in response to thiamine limitation, helping researchers understand regulatory feedback mechanisms.

• Subcellular Localization Studies: Immunofluorescence microscopy using thiaminase-1 antibodies can reveal changes in enzyme localization under thiamine stress conditions, potentially identifying novel functional compartmentalization.

• Protein-Protein Interaction Analysis: Co-immunoprecipitation experiments with thiaminase-1 antibodies can identify interaction partners that may change during thiamine deficiency, revealing regulatory networks.

• Stress Response Pathway Investigation: As demonstrated in breast cancer research, thiaminase-1 exposure induces ER stress response proteins like GRP78 . Antibodies can be used to track such stress response markers in relation to thiaminase-1 activity or inhibition.

• Cell Type-Specific Vulnerability Assessment: Immunohistochemistry with thiaminase-1 antibodies in tissues under thiamine deficiency can reveal differential expression patterns across cell types, helping identify particularly vulnerable populations.

Research has shown that thiaminase-1 exposure triggers the unfolded protein response in breast cancer cell lines, suggesting that antibodies against both thiaminase-1 and stress response proteins can provide comprehensive insights into cellular adaptation mechanisms under thiamine stress conditions .

What role might thiaminase-1 play in cancer biology and potential therapeutic applications?

Research has revealed intriguing connections between thiaminase-1 and cancer biology with potential therapeutic implications:

• Exploiting Nutritional Vulnerability: Studies have demonstrated that breast cancer cell lines show significant sensitivity to recombinant thiaminase-1 enzyme, with IC50 values ranging from 0.012 to 0.022 U/ml . This sensitivity appears linked to the downregulation of thiamine transporter THTR2 in breast cancer cells, creating a nutritional vulnerability that could be therapeutically exploited .

• Synergistic Interactions with Chemotherapeutics: Research has shown that thiaminase-1 demonstrates synergistic or additive effects when combined with conventional chemotherapeutic agents:

  Cell Line	Thiaminase-1 + Doxorubicin	Thiaminase-1 + Paclitaxel
  MCF-7	Synergistic (CI<0.8)	Synergistic (CI<0.8)
  ZR75	Synergistic (CI<0.8)	Synergistic (CI<0.8)
  HS578T	Synergistic (CI<0.8)	Synergistic (CI<0.8)
  T47D	Synergistic (CI<0.8)	Synergistic (CI<0.8)

  Note: CI (Combination Index) values below 0.8 indicate synergistic interactions

• Metabolic Stress Induction: Thiaminase-1 may function as a metabolic stressor in cancer cells, potentially triggering the unfolded protein response (UPR) as evidenced by increased expression of GRP78, without activating energy-sensing kinases like AKT, AMPK, and GSK-3β .

• In Vivo Tumor Growth Inhibition: Xenograft studies with MDA231 breast cancer cells demonstrated that thiamine starvation induced tumor growth delay, suggesting potential therapeutic relevance .

Thiaminase-1 antibodies are valuable tools for investigating these phenomena, enabling researchers to track enzyme distribution in tumors, monitor therapeutic responses, and understand resistance mechanisms in preclinical models.

What are the technical challenges in developing highly specific antibodies against thiaminase-1?

Developing highly specific antibodies against thiaminase-1 presents several technical challenges that researchers must address:

• Limited Evolutionary Conservation: Thiaminase-1 shows variable sequence conservation across species, particularly between bacterial sources (like B. thailandensis or B. thiaminolyticus) and potential eukaryotic versions. This necessitates careful epitope selection for antibody development.

• Cross-Reactivity Concerns: Given that thiaminase-1 cleaves thiamine, there may be structural similarities with other thiamine-binding proteins or metabolic enzymes, creating potential for cross-reactivity. Extensive validation against related proteins is essential.

• Native Conformation Preservation: For applications requiring recognition of the native enzyme (such as immunoprecipitation or functional blocking studies), antibodies must recognize properly folded thiaminase-1, which may differ from denatured forms used in some immunization protocols.

• Variable Post-Translational Modifications: If thiaminase-1 undergoes post-translational modifications in different contexts, antibodies raised against the unmodified recombinant protein may show variable recognition of native forms.

• Detection in Complex Samples: When thiaminase-1 is present at low abundance in complex biological samples, sensitivity becomes critical. Strategies such as using detection systems with signal amplification or developing sandwich ELISA approaches may be necessary.

To address these challenges, researchers should consider developing panels of monoclonal antibodies targeting different epitopes, extensive validation across multiple assay platforms, and confirmation of specificity using tissues or samples from thiaminase-1 knockout models.

How does thiaminase-1 contribute to microbial survival and competitive advantage?

Research has revealed that thiaminase-1 provides bacteria with distinct metabolic advantages:

• Precursor Salvage Mechanism: In the soil bacterium B. thailandensis, thiaminase-1 facilitates the salvage of valuable thiamine precursors (thiazole and hydroxymethyl pyrimidine) from environmental thiamine . This salvage pathway provides a growth advantage to thiamine auxotrophs that cannot synthesize thiamine de novo but can utilize these precursors.

• Growth Advantage Under Thiamine Limitation: Experimental evidence demonstrates that thiaminase-1 extends the survival of thiamine auxotrophic bacterial strains when grown in defined media with limited thiamine availability . This suggests a role in nutrient scavenging during competitive growth conditions.

• Utilization of Phosphorylated Thiamine Forms: Thiaminase-1 can cleave not only thiamine but also its phosphorylated derivatives, enabling bacteria to access precursors from various thiamine-containing compounds in the environment .

• Detoxification of Thiamine Analogs: Research suggests thiaminase-1 may help bacteria detoxify and recycle components from certain thiamine analogs that might otherwise be harmful, providing an additional survival advantage .

• Ecological Interaction Mediation: Thiamine and its precursors serve as drivers of ecological interactions between microorganisms, with thiaminase-1 potentially playing a role in this complex interplay by modulating the availability of these compounds in microbial communities .

These findings establish thiaminase-1 as a multifunctional enzyme that contributes to microbial fitness through nutrient acquisition strategies, potentially explaining its conservation in certain bacterial lineages despite its seemingly counterintuitive function of destroying an essential vitamin.

What cellular pathways are affected by thiaminase-1 activity in mammalian systems?

In mammalian systems, thiaminase-1 activity impacts several critical cellular pathways through its effects on thiamine availability:

Understanding these pathway interactions is critical for researchers investigating both the pathological consequences of thiaminase-1 exposure and its potential therapeutic applications in contexts like cancer treatment.

How might gene expression analysis complement antibody-based detection of thiaminase-1?

Gene expression analysis provides complementary insights to antibody-based detection of thiaminase-1, offering several distinct advantages:

• Transcriptional Regulation Insights: While antibodies detect protein presence, gene expression analysis reveals transcriptional regulation patterns of the thiaminase-1 gene (proposed name thiA) . This allows researchers to identify regulatory factors and conditions that influence thiaminase-1 production at the mRNA level.

• Temporal Resolution of Expression Dynamics: Gene expression analysis can capture rapid changes in thiaminase-1 transcription before protein levels change, providing better temporal resolution of expression dynamics during environmental shifts or stress responses.

• Detection in Challenging Samples: In samples where protein extraction is difficult or antibody recognition is compromised (fixed tissues, certain organisms), mRNA detection may provide more reliable detection of thiaminase-1 presence.

• Comparative Expression Profiling: RNA-seq or microarray approaches allow simultaneous analysis of thiaminase-1 expression alongside other genes, revealing co-expression patterns that may indicate functional relationships or shared regulatory mechanisms.

• Species Identification in Mixed Samples: In complex microbial communities, gene sequence analysis can distinguish thiaminase-1 from different bacterial sources, whereas antibodies might not differentiate between orthologs with similar epitopes.

• Mutational Analysis: Gene sequencing can identify variants or mutations in the thiaminase-1 gene that might affect enzyme function but not necessarily antibody recognition, providing additional functional information.

For comprehensive research approaches, combining antibody-based protein detection with gene expression analysis provides the most complete picture of thiaminase-1 biology, revealing both transcriptional control mechanisms and the resulting protein expression patterns.

What are the optimal storage and handling conditions for thiaminase-1 antibodies to maintain their specificity and sensitivity?

Proper storage and handling of thiaminase-1 antibodies is critical for maintaining their specificity and sensitivity across experimental applications:

• Storage Temperature:

  • Primary storage: Maintain antibodies at -20°C for long-term storage

  • Working aliquots: Store at 4°C for up to one month to avoid repeated freeze-thaw cycles

  • Avoid storing at room temperature for extended periods

• Aliquoting Strategy:

  • Upon receipt, divide antibody solutions into single-use aliquots

  • Use small volume aliquots (10-50 μl) to minimize freeze-thaw cycles

  • Include carrier proteins (0.1-1% BSA) in dilute antibody solutions

• Buffer Considerations:

  • For long-term storage, phosphate-buffered solutions at pH 7.2-7.6 with preservatives are recommended

  • Avoid sodium azide when using antibodies with HRP-conjugated detection systems

  • Consider adding glycerol (30-50%) to prevent freeze-thaw damage

• Contamination Prevention:

  • Use sterile technique when handling antibody solutions

  • Include antimicrobial preservatives in working solutions

  • Filter solutions if precipitates form

• Stability Testing Protocol:

  • Periodically test antibody performance using consistent positive controls

  • Document lot-specific performance metrics for longitudinal comparison

  • Consider running parallel tests with new and older antibody lots

• Shipping and Transport:

  • Transport on ice or with cold packs for short distances

  • Use dry ice for longer shipments

  • Avoid prolonged exposure to ambient temperatures

Maintaining detailed records of antibody performance over time allows researchers to identify potential degradation issues early and ensure experimental reproducibility across studies.

What are the most effective experimental designs to study thiaminase-1 inhibition in research models?

Effective experimental designs for studying thiaminase-1 inhibition require careful consideration of several key elements:

• In Vitro Enzyme Inhibition Studies:

  • Use purified recombinant thiaminase-1 with spectrophotometric assays

  • Screen potential inhibitors at multiple concentrations to generate dose-response curves

  • Include appropriate positive controls (known inhibitors if available) and negative controls

  • Determine inhibition kinetics (competitive, non-competitive, uncompetitive) through Lineweaver-Burk or similar analyses

• Cellular Models:

  • Select cell lines expressing thiaminase-1 or transfect with thiaminase-1 expression constructs

  • Compare thiaminase-1 activity in treated versus untreated cells using activity assays

  • Monitor cellular consequences of inhibition (growth, metabolism, stress responses)

  • Use thiaminase-1 antibodies to assess whether inhibitors affect protein levels or just activity

• Bacterial Studies:

  • Compare growth of thiaminase-1-producing bacterial strains (like B. thailandensis) with and without inhibitors under thiamine-limited conditions

  • Include thiaminase-1 knockout strains as negative controls

  • Use serial dilution survival assays similar to those employed in thiamine auxotroph studies

• Animal Models:

  • Design studies with thiaminase-1-sensitive models (e.g., cancer xenografts shown to respond to thiamine starvation)

  • Monitor both target inhibition (using antibodies to assess thiaminase-1 activity/levels) and physiological outcomes

  • Include appropriate dosing schedules and administration routes based on inhibitor properties

• Combination Approaches:

  • Test thiaminase-1 inhibitors in combination with other relevant treatments

  • For cancer studies, assess potential synergies with chemotherapeutics, similar to the thiaminase-1 enzyme synergy studies with doxorubicin and paclitaxel

• Biomarker Analysis:

  • Monitor downstream effects of thiaminase-1 inhibition using relevant biomarkers

  • For example, measure GRP78 expression changes as an indicator of ER stress response modulation

These experimental designs should incorporate appropriate controls, statistical analyses, and validation approaches to ensure robust and reproducible results.

How can researchers troubleshoot inconsistent results when using thiaminase-1 antibodies in different experimental contexts?

When facing inconsistent results with thiaminase-1 antibodies across different experimental contexts, researchers should implement a systematic troubleshooting approach:

• Antibody Validation Assessment:

  • Verify antibody specificity using positive controls (recombinant thiaminase-1, bacteria known to express the enzyme)

  • Test against negative controls (thiaminase-1 knockout samples if available)

  • Perform epitope mapping or competition assays to confirm binding specificity

  • Consider testing multiple antibodies targeting different epitopes

• Sample Preparation Variables:

  • Evaluate different protein extraction methods (RIPA vs. gentler lysis buffers)

  • Test various fixation protocols for immunohistochemistry applications

  • Assess whether sample processing affects thiaminase-1 epitope accessibility

  • Consider native vs. denaturing conditions for different applications

• Technical Optimization:

  • Titrate antibody concentrations across a wider range than normally used

  • Modify blocking agents to reduce background (consider milk vs. BSA vs. serum)

  • Adjust incubation conditions (time, temperature, buffer composition)

  • For Western blots, try different transfer methods (wet vs. semi-dry)

• Context-Specific Considerations:

  • When working with bacteria, consider growth phase effects on thiaminase-1 expression as demonstrated in B. thailandensis studies showing peak activity at different timepoints

  • For mammalian studies, assess whether cellular stress conditions might alter epitope accessibility or protein localization

  • Consider species-specific differences in thiaminase-1 structure that might affect antibody recognition

• Biological Variables Tracking:

  • Document experimental conditions thoroughly (cell density, passage number, culture media composition)

  • Monitor thiamine levels in experimental systems as they may affect thiaminase-1 expression

  • Track bacterial growth phases carefully as thiaminase-1 activity peaks at specific timepoints in culture

• Complementary Method Validation:

  • Confirm protein presence using mass spectrometry when antibody results are questionable

  • Verify gene expression using RT-PCR or RNA-seq approaches

  • Assess enzyme activity using functional assays to complement antibody-based detection

By systematically addressing these factors, researchers can identify sources of variability and establish reliable protocols for consistent thiaminase-1 antibody performance across experimental contexts.

What emerging technologies might enhance the study of thiaminase-1 in complex biological systems?

Several cutting-edge technologies offer promising approaches to advance thiaminase-1 research in complex biological systems:

• CRISPR-Cas9 Gene Editing:

  • Creation of precise thiaminase-1 knockout models across diverse organisms

  • Generation of epitope-tagged endogenous thiaminase-1 for improved detection

  • Development of conditional expression systems to study temporal aspects of thiaminase-1 function

• Single-Cell Analysis Techniques:

  • Single-cell RNA sequencing to identify cell-specific thiaminase-1 expression patterns

  • Mass cytometry (CyTOF) with thiaminase-1 antibodies to understand expression heterogeneity

  • Spatial transcriptomics to map thiaminase-1 expression in tissue contexts

• Advanced Imaging Approaches:

  • Super-resolution microscopy for detailed subcellular localization studies

  • Intravital microscopy to track thiaminase-1 activity in living organisms

  • FRET-based biosensors to monitor thiaminase-1 activity in real-time

• Proximity Labeling Technologies:

  • BioID or APEX2 fusion proteins to identify thiaminase-1 interaction partners

  • Spatial proteomics to understand thiaminase-1's position within cellular protein networks

  • Interactome mapping under various thiamine availability conditions

• Metabolomic Integration:

  • Untargeted metabolomics to comprehensively profile metabolic changes induced by thiaminase-1

  • Stable isotope tracing to track thiamine metabolism and precursor salvage

  • Integration of metabolomic and transcriptomic data for systems-level understanding

• Microbiome Analysis Tools:

  • Metagenomic sequencing to identify thiaminase-1-producing organisms in complex communities

  • Functional metagenomics to discover novel thiaminase variants

  • Gnotobiotic models to study thiaminase-1's role in host-microbiome interactions

These emerging technologies, when applied to thiaminase-1 research, promise to reveal new insights into this enzyme's biological functions, regulatory mechanisms, and potential applications in both basic science and therapeutic contexts.

How might structural biology approaches contribute to understanding thiaminase-1 function and developing specific inhibitors?

Structural biology approaches offer powerful tools for elucidating thiaminase-1 function and facilitating inhibitor development:

• High-Resolution Structure Determination:

  • X-ray crystallography of thiaminase-1 with various substrates and cofactors

  • Cryo-electron microscopy for visualizing thiaminase-1 in different conformational states

  • NMR spectroscopy to capture dynamic aspects of enzyme function

• Structure-Function Analysis:

  • Identification of catalytic residues through site-directed mutagenesis guided by structural data

  • Comparison of thiaminase-1 structures across species to identify conserved functional domains

  • Elucidation of structural changes during substrate binding and catalysis

• Rational Inhibitor Design:

  • Structure-based virtual screening of compound libraries against the thiaminase-1 active site

  • Fragment-based drug discovery to identify building blocks for inhibitor development

  • Computational modeling of protein-ligand interactions to predict binding affinities

• Protein Engineering Approaches:

  • Creation of modified thiaminase-1 variants with altered substrate specificity

  • Development of catalytically inactive mutants for research applications

  • Engineering of thiaminase-1 fusion proteins for targeted delivery in therapeutic applications

• Biophysical Characterization:

  • Isothermal titration calorimetry to quantify binding thermodynamics

  • Surface plasmon resonance for real-time binding kinetics analysis

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

• Comparative Structural Analysis:

  • Structural comparison with other thiamine-binding proteins to understand specificity determinants

  • Analysis of differences between bacterial and potential eukaryotic thiaminase-1 structures

  • Identification of allosteric sites that might offer alternative inhibition strategies

These structural biology approaches would significantly advance understanding of thiaminase-1 function while providing essential information for developing specific inhibitors with potential research and therapeutic applications.

What are the potential applications of thiaminase-1 antibodies in developing diagnostic tools for thiamine-related disorders?

Thiaminase-1 antibodies hold significant potential for developing diagnostic tools for thiamine-related disorders through several innovative approaches:

• Detection of Thiaminase-1-Producing Microorganisms:

  • Development of immunoassays to identify thiaminase-1-producing bacteria in clinical samples

  • Immunohistochemical techniques to locate these organisms in tissue biopsies

  • Creating rapid diagnostic tests for clinical settings where thiaminase-1-induced deficiency is suspected

• Monitoring Thiaminase-1 Exposure:

  • ELISA-based testing to detect thiaminase-1 in food samples associated with thiamine deficiency

  • Analysis of thiaminase-1 levels in gastrointestinal contents to assess potential dietary exposure

  • Measurement of anti-thiaminase-1 antibodies in patients as markers of previous exposure

• Cancer Diagnostics Applications:

  • Assessment of thiamine transporter (THTR2) and thiaminase-1 sensitivity profiles in tumor biopsies

  • Development of companion diagnostics for potential thiaminase-1-based therapies

  • Monitoring thiaminase-1 activity in patients receiving experimental therapies

• Precision Medicine Approaches:

  • Stratification of patients with suspected thiamine deficiency based on thiaminase-1 exposure

  • Identification of individuals who might benefit from specific anti-thiaminase interventions

  • Personalized nutritional recommendations based on thiaminase-1 risk assessment

• Point-of-Care Testing Development:

  • Creation of lateral flow immunoassays for rapid thiaminase-1 detection in field settings

  • Development of miniaturized detection platforms for resource-limited environments

  • Integration with smartphone-based analysis for wider accessibility

• Environmental Monitoring:

  • Testing water sources for thiaminase-1-producing microorganisms in regions with high thiamine deficiency prevalence

  • Monitoring thiaminase-1 activity in food production systems where thiamine deficiency is a concern

These diagnostic applications would require rigorous validation against established methods of assessing thiamine status, but offer the potential to identify specific causes of thiamine deficiency and guide more targeted interventions in both clinical and public health contexts.