An updated transketolase activity assay to probe thiamine utilization in fish

Abstract

Thiamine (vitamin B1) deficiency is causative of reproductive failures and population declines in lake trout, Atlantic salmon, and other predatory fish species. Transketolase (TKT) is the rate-limiting enzyme of the non-oxidative phase of the pentose-phosphate pathway. TKT is critical for mediating the availability of sugars to return to glycolysis and for synthesizing NADPH and R5P, which are needed to maintain the cellular oxidation state and to produce biomolecules necessary for successful growth and reproduction. TKT activity provides a measure of functional thiamine availability since it requires thiamine diphosphate (TDP) as a coenzyme. Its activity is usually analyzed via a coupled enzyme reaction, including ribose-5-phosphate (R5P) and xylulose-5-phosphate (X5P) as substrates to permit the kinetic monitoring of the depletion of exogenous NADH. We developed a simplified, cost-effective procedure for quantifying TKT activity in fish liver to probe thiamine utilization and magnesium (Mg²⁺) dependence. Unlike previous protocols, the method omits costly X5P, relying instead on endogenous enzyme activity for in situ substrate generation. In two lake trout strains, TKT-specific activity correlated with TDP concentration while maximal activity reflected enzyme abundance and holoenzyme stability. By running samples with and without Mg²⁺ and over a range of TDP concentrations, the assay framework allows for distinguishing a Mg²⁺ limitation from a thiamine limitation and defines apparent EC₅₀ and V_max values. This simplified and tunable assay provides a tool for evaluation of thiamine-related metabolic resistance under dietary or environmental stress across fish populations and species.