Thiamine pyrophosphate assay

Talwar D, Davidson H, Cooney J, St JO Reilly D. Vitamin B(l) status assessed by direct measurement of thiamin pyrophosphate in erythrocytes or whole blood by HPLC comparison with erythrocyte transketolase activation assay. Clin Chem 2000 46 704-10. 

Thiamin (vitamin Bi) Thiamin in the body is chiefly found in the phosphorylated form thiamin pyrophosphate (TPP) which is a coenzyme. The majority (80%) of thiamin in the blood is found in the erythrocytes and assay of blood thiamin is a more reliable indicator of deficiency than assay of erythrocyte transketolase. The phosphorylated vitamers are enzymically converted to thiamin in samples using diastase following deproteinization. To reach the low picomolar concentrations the thiamin compounds are oxidized by ferricyanide to form thiochromes, which are highly fluorescent. The thiochromes are then separated by reversed-phase HPLC and detected by their emission at 425-450 nm. 

A more specific type of chemical assay is based on enzymatic measurement of vitamin co-enzyme activity. This approach is designed to detect a vitamin deficiency in tissues, and is only feasible for those vitamins that serve as co-enzymes. For instance, thiamin depletion in a subject can be diagnosed by measuring the transketolase activity in red blood cells with and without the addition of thiamin pyrophosphate (TPP) in vitro. If TPP increases the activity by more than a given amount, thiamin deficiency is indicated. Similarly, a subnormal level of riboflavin is indicated in tissues if the activity of erythrocyte glutathione reductase is increased after the addition of flavin adenine dinucleotide (FAD). Erythrocyte transaminase activation by pyridoxal-5 -phosphate (PLP) can be measured to establish a deficiency of vitamin B . 

Abbreviations TPP, thiamin pyrophosphate MNA, A/ -methyInicotinamide 2-Pyr, A/ -methyl-2-pyridone-5-carboxamide PLP, pyridoxal-5 -phosphate LC, liquid chromatography UV, ultraviolet detection FL, fluorescence detection CPB, competitive proteinbinding assay. 

Nutritional status assessment for thiamine is generally carried out by assaying the total thiamine in whole blood or erythrocytes, or by measuring the activity of erythrocyte transketolase before and after incubation with exogenous thiamine pyrophosphate. The latter serves as the sensitive index of thiamine nutritional status (Brin 1980). In addition to the enzymatic test, a measure of urinary thiamine in relation to dietary intake has been the basis for balance studies to assess the adequacy of intake. When thiamine excretion is low, a larger portion of the test dose is retained, indicating a tissue s need for thiamine. A high excretion indicates tissue saturation. In the deficient state, excretion drops to zero. Plasma pyruvate and lactate concentrations have also been used to assess thiamine status. 

Strictly specific for thiamine are the yeast Kloeckera brevis and the bacteria Lactobacillus fermentum 36 and Lactobacillus viridescens. The first will measure 0.2-2.0 m g of thiamine, the two latter 5-40 m/ g. They all also respond to thiamine pyrophosphate, but to a different degree therefore the material under assay should be hydrolysed by a phosphatase preparation prior to assay. 

Thiamine pyrophosphate is required as a cofactor for transketolase activity. Decreased erythrocyte transketolase activity is therefore found in thiamine deficiency and activity is increased by the addition of thiamine pyrophosphate to the assay system. 

Because the basic biological fimction of thiamine is to act as the pyrophosphate cofactor in a number of enzyme systems, two differing approaches to assessment of status became available. The analyte, either free or phosphorylated, can be measured directly in a suitable body fluid or tissue or its properties as an enzymatic cofactor can be exploited in a functional assay. Both approaches have their advantages and... 

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