Transketolase assessment

Other ThDP enzymes catalyze the formation of C-C bonds by transferring C2 units from a donor compound to an acceptor compound (transferase activity). We have studied the E. coli transketolase with respect to structure-function relationships, as well as to possible applications in asymmetric syntheses (Section 2.2.2.2.1). During the late 1990s a transketolase-like enzyme, 1-deoxyxylulose 5-phosphate synthase, was discovered. Its structure and value in chemoenzymatic syntheses were also assessed in project B21 (Section 2.2.2.2.2). 

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%. 

Determination of the effective functioning of particular enzymes or metabolic pathways potentially may be useful in demonstrating adequacy of provision. Enzymes in plasma that may be helpful in this regard are glutathione peroxidase as an index of selenium status, and red cell enzymes, such as transketolase (thiamine), glutathione reductase (riboflavin) or transaminase (pyridoxine), or glutathione peroxidase (selenium) are all widely used. Methyltetrahydrofolate reductase is involved in metabolism of homocysteine, hence assessment of plasma homocysteine is a useful measure of... 

As there are no reports of adverse effects from consumption of excess thiamine from food and supplements (supplements of 50 mg/day are widely available without prescription), and the data are inadequate for a quantitative risk assessment, no UL has been defined for thiamine. However, as stimulators of transketolase enzyme synthesis such as thiamine support a high rate of nucleic acid ribose synthesis necessary for tumor cell survival, chemotherapy resistance, and proliferation, some concern has been expressed that thiamine supplementation of common food products may contribute to increased cancer rates in the Western world. There is, however, littie evidence to support this assumption. Rarely, individuals given high-dose intravenous thiamine in treatment of beriberi have developed anaphylaxis, the frequency being about 1 100,000. 

TABLE 30-3 Relative Me Thiamine Pyrophosphate) Transketolase) Measuremc Tiiiamine Status rits 6Tp i recT E ryth rp Cyte o 1 Fu n c ti o nal i (Eryrh rocyte nts in Assessing... 

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 can be assessed by measuring blood levels. Increased blood levels of pyruvate and lactate suggest thiamine deficiency. Measurement of erythrocyte transketolase activity, which requires TPP as a coenzyme, confirms the deficiency. 

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. 

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... 

ETK based methods, once considered the most reliable means of assessing thiamine status, are now considered inadequate because they only provide an indirect measure. Because transketolase activity requires thiamine, decreased transketolase activity is presumed to be due to a decrease in thiamine. However, other factors may decrease transketolase activity including decreased enzymatic binding and decreased enzyme synthesis as has been demonstrated in diseases such as diabetes (Friedrich 1988) and liver dysfunction (Feimelly et al. 1967). ETK based methods have also been criticized as unreliable, insensitive, and subject to poor precision (Bailey et al. 1994). 

Erythrocyte transketolase activity was the classic method to assess thiamine status. Two samples of blood are incubated with excess substrate for the pentose phosphate pathway to one is also added excess thiamine diphosphate while the other serves as the control. The amount of substrate remaining and product formed are quantified, and any enhancement in activity resulting from the added thiamine diphosphate indicates that the sample was originally deficient in thiamine to some extent. 

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. 

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. 

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. 

In general, the recommended allowances are based (1) on assessments of the effects of varying levels of dietary thiamin on the occurrence of clinical signs of deficiency, (2) on the excretion of thiamin or its metabolites, and (3) on erythrocyte transketolase activity. Most studies have been conducted on subjects fed diets with ratios of carbohydrate and fatsimilar to those commonly consumed in the United States. There is evidence that dietary fat spares thiamin to some extent... 

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