Reassessing Transketolase Assays: Methodological Considerations for Detecting Functional Thiamine Deficiency.

Thiamine (vitamin B1) deficiency due to degradation, inadequate intake, impaired absorption, or increased metabolic demand remains prevalent in both human and animal populations. In its diphosphate form (thiamine diphosphate, TDP), thiamine serves as an essential cofactor for metabolic enzymes, including transketolase (TKT), a rate-limiting enzyme of the pentose-phosphate pathway. Measurement of TKT activity with and without exogenous TDP provides a functional assessment of thiamine utilization that serves as a surrogate for or complements direct quantification of thiamine forms. Beyond its historical role as a deficiency-screening tool, the TKT assay offers opportunities to interrogate enzyme function and cofactor dependence. Strategic variation of assay conditions, such as higher TDP concentrations to identify low-affinity TKT variants or including or omitting magnesium to assess functional cofactor limitation, can distinguish true deficiency from impaired enzyme utilization or altered enzyme properties. This review evaluates preanalytical variables, assay methodologies, and data presentation strategies used for TKT measurements in erythrocytes and other tissues across human and nonhuman studies. Emphasis is placed on biological and methodological determinants that shape measured activity and responsiveness. Improved interpretation frameworks and thoughtful assay design can expand the utility of TKT measurements as indicators of functional thiamine status across clinical, translational, and ecological contexts.