Transketolase assays

Fluorogenic compound (56) for transketolase assays has been prepared making use of FruA specificity [123]. Pendant anionically charged chains have been extended from O- or C-glycosidic aldehydes to furnish low molecular weight mimics of the sialyl Lewis X tetrasaccharide such as (SS) (Figure 10.23) [124], Other higher carbon... 

Smeets EH, Muller H, de Wael J. A NADH-dependent transketolase assay in erythrocyte hemolysates. Clin Chim Acta 1971 33 379-86. 

Boni, L., Kieckens, L., and Hendrikx, A., 1980. An evaluation of a modified erythrocyte transketolase assay for assessing thiamine nutritional adequacy. Journal of Nutrition Science and Vitaminology. 26 507-514. 

However, there are no clear cut-off values for basic laboratory parameters, which could be useful for preselecting patients at risk of potential detrimental conditions. Some parameters that are easy to detect, such as lactic/pyruvic metabolic acidosis, are not specific for thiamine deficiency (Table 33.2). On the other hand, blood TDP and transketolase assays have low sensitivity due to significant overlapping results between healthy and thiamine-deficient individuals. This makes laboratory assessment of borderline thiamine deficiency conditions difficult. Thus, anamnesis and awareness of the socio-demographic conditions of the patient are as important as laboratory tests in the early diagnosis of thiamine deficiency. 

Protein was determined by the method of Lowry, using a calibration curve which had been obtained with a TK solution standardized by the Biuret method. TPP was determined by radioactivity measurements. The transketolase assay was carried out in the presence and absence of added thiamine diphosphate/Mg++. A stimulation of 13 % of enzymatic activity was found when the coenzyme was added, indicating a loss of bound coenzyme upon gel filtration. Assuming a linear relationship between enzymatic activity and thiamine diphosphate molecules bound to apotransketolase the value found for thiamine diphosphate was corrected by these 13 %. 

TALDO deficiency can be confirmed in lymphoblasts, fibroblasts and in erythrocytes. These cells are incubated with ribose-5-phosphate, after which formation of transketolase and TALDO products are analysed by gas chromatography with nitrogen phosphorous detection by liquid chromatography tandem mass spectrometry [8, 11]. A similar enzyme assay is available for RPI [2]. Confirmation of the gene defect can be performed by sequence analysis. Disease-causing mutations have been detected in all TALDO-deficient patients and in the RPI-deficient patient. 

Sevestre, A., Helaine, V., Guyot, G., Martin, C. and Hecquet, L. (2003) A fluorogenic assay for transketolase from... 

Figure 18.12 Enzymatic assay for transketolase based on L-tyrosine release.

In the first step, we showed by analytical studies that compound 28 was a donor substrate for transketolase in the presence of D-ribose-5-phosphate as acceptor substrate and that in the second step, the hydroxylated aldehyde released 29 led to the P-elimination of protected L-tyrosine. We showed that the free L-tyrosine can thus be obtained by enzymatic deprotection of N-acetyl-L-tyrosine ethyl ester using acylase and subtilisine. In this conditions, it should be possible to carry out this assay in vivo in the presence of host cells overexpressing transketolase and auxotrophic for L-tyrosin. This strategy should offer the first stereospecific selection test of transketolase mutants. The principle of this assay may be extended to other enzymes that can release aldehydes P-substituted by L-tyrosine. 

The most reliable method for assessing thiamin status involves the measurement of red blood cell transketolase. This enzyme is measured with and without the addition of TPP to the enzyme assay mixtures. In dietary thiamin deficiency, synthesis of transketolasc continues, but conversion of the apoet zyme to the holoenzyme in the cell is inhibited, resulting in the accumulation of the enzyme in the apoenzyme form. Addition of TPP to cell homogenates results in the conversion of apoenzyme to holoenzyme. This conversion can easily be detected by enzyme assays. The amount of shmulation of enzyme activity by the added TPP is used to assess thiamin status. A deficiency is indicated by a shmulation of over 20%, The TPP-dependent stimulation, using red blood cells from normal subjects, ranges from 0 to 15%. 

FIGURE 9.72 Stimulatory effect of adding thiamin triphosphate (TPP) during assays of red blood cell transketolase, using blood samples taken during the consumption of different diets. (Redrawn with permission from Ariaey-Nejad et al, 1970.)... 

The most commonly used enzyme for the functional assay is transketolase. Transketolase catalyzes two reactions in the pentose phosphate pathway (Figure 30-10). As an enzyme within the erythrocyte, transketolase is independent of nonspecific changes in the extracellular plasma. As vitamin Bi deficiency becomes more severe, (1) thiamine becomes limiting in the body cells, (2) the amount of the coenzyme is depleted, and (3) the transketolase activity sub-... 

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. 

Thiamine deficiency is most frequently assessed by assaying erythrocyte transketolase activity in the presence and absence of added TPP. If the red blood cells have sufficient thiamine, the transketolase will be fully saturated with TPP, and no increase in activity will be observed when TPP is added to the assay system. An increase in transketolase activity indicates that the patient is thiamine deficient. 

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. 

Standard methods for assessment of thiamine status used to be determination of erythrocyte transketolase (a-ETK) activity (EC 2.2.1.1) with and without stimulation of this enzyme by addition of TDP cofactor (TOP TK effect). A TDP TK effect >15% is considered to show some degree of deficiency, whereas values >22% are considered to indicate severe deficiency. Technical difficulties, including standardization of the assay, instability of the enzyme during storage, and various conditions possibly influencing apoenzyme concentrations led to an increasing use of direct determination of TDP in whole blood, e.g., by HPLC in order to assess thiamine status. The HPLC assay is more robust and easier to perform. Thiamine... 

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 . 

This is an enzyme of the pentose phosphate shunt, which may be assayed by u.v. spectroscopy (together with transketolase) by using an NAD coupled reaction [618]. 

Frost and Draths (23) were unsuccessful in their attempt to continue their systematic accumulation of intermediates in the aromatic amino acid biosynthetic pathway when the three enzymes discussed above (DAHP synthase, transketolase, and DHQ synthase) were overexpressed in an E. coli strain lacking chorismate synthase activity. This led to the discovery that under the conditions of their assay, catechol was being produced, along with beta-ketoadipate. The Klebsiella pneumoniae enzymes involved in the conversion of DHS to catechol, DHS dehydratase and protocatechuate decarboxylase, were used since the corresponding E. coli enzymes have not yet been cloned. This discovery may have a significant industrial impact, as these two chemicals have important uses in the chemical industry. Catechol, for example, can be used to produce the flavoring vanillin, as well as L-dopa, epinephrine, and norepinephrine. Adipic acid is used in the production of nylon-6,6. 

The erythrocyte transketolase (ETK) activation assay (also known as the saturation test) measures the functional capacity of the enzyme transketolase in red blood cells i.e. erythrocytes). Transketolase is a thiamine-dependent enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), a process of glucose turnover that produces nicotinamide adenine dinucleotide phosphates (NADPH) as reducing equivalents and pentose sugars as essential components of nucleotides. In the absence of adequate thiamine, the PPP output is compromised. 

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. 

Cascade ReactionsJbr Assaying Transketolase Activity In Vitro I 317... 

Cascade Reactions for Assaying Transketolase Activity by In Vivo Selection J29... 

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