Erythrocyte transketolase

These various reports stress the need to supplement parenteral nutrition with thiamine-containing vitamins unless there is adequate dietary intake, and to monitor serum thiamine and erythrocyte transketolase activity so that supplementary thiamine can be given in good time, if necessary intravenously (45). Giving thiamine will not rectify the various disorders if hepatic function is severely disturbed, because then thiamine is not phosphorylated and hence remains physiologically inactive. 

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-... 

Baines M, Davies G. The evaluation of erythrocyte thiamin diphosphate as an indicator of thiamin status in man, and its comparison with erythrocyte transketolase activity measurements. Ann Clin Biochem 1988 25 (Pt 6) 698-705. 

Puxty JA, Haskew AE, Ratcliffe JG, McMurray J. Changes in erythrocyte transketolase activity and the thiamine pyrophosphate effect during storage of blood. Ann Clin Biochem 1985 22 (Pt 4) 423-7. 

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. 

Elderly people living on their own frequently have an inadequate diet. This is particularly true of men if they are unused to cooking for themselves. This patient may have a number of micronutrient deficiencies but, acutely, the most important would be possible thiamine deficiency. This can be detected by demonstrating an increase in the percentage activation of erythrocyte transketolase in vitro by the addition of thiamine or the measurement of thiamine pyrophosphate in erythrocytes. 

Kaufmann, A., Uhlhaas, S., Friedl, W, and Propping, P. (1987). Human erythrocyte transketolase no evidence for variants. Clin. Chim. Acta. 162 215-219. 

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... 

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. 

ETK AC = Erythrocyte Transketolase Activity Coefficient TDP = thiamine diphosphate N.S. = not specified MTD = moderate thiamine deficiency TD = thiamine... 

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. 

Bailey, A.L., Finglas, P.M., Wright, A.J., and Southon, S., 1994. Thiamin intake, erythrocyte transketolase (EC 2.2.1.1) activity and total erythrocyte thiamin in adolescents. British Journal of Nutrition. 72 111-125. 

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. 

Brin, M., 1962. Erythrocyte transketolase in early thiamine deficiency. Annals of the New York Academy of Sciences. 98 528-541. 

McLaren, D.S., Docherty, M.A., and Boyd, D.H., 1981. Plasma thiamin pyrophosphate and erythrocyte transketolase in chronic alcoholism. The American Journal of Clinincal Nutrition. 34 1031-1033. 

Warnock, L.G., Prudhomme, C.R., and Wagner, C., 1978. The determination of thiamin pyrophosphate in blood and other tissues, and its correlation with erythrocyte transketolase activity. The Journal of Nutrition. 108 421-427. 

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. 

Erythrocyte transketolase activity was found to be lowered in different groups of TD patients (Herve et al. 1995 Khounnorath et al. 2011) (Table 33.2). However, post mortem brains of alcoholics who died without symptoms of Wernicke s encephalopathy (WE) revealed 20-35% reductions in the activities of all TDP-dependent enzymes in various brain regions (Lavoie and Butterworth 1995). This means that some degree of TD-induced reductions in oxidative metabolism may be tolerated. In fact, individual sensitivity to TDP deficits may be modified by coexisting clinical conditions such as alcoholism, renal insufficiency, dialysis programmes, aging, diabetes, cardiovascular complications, and voluntary or socio-economically dependent dietary habits (Tables 33.1 and 33.2). 

ETK, erythrocyte transketolase activity TDP, thiamine diphosphate ETK-A-TDP, activation... 

There are laboratory tests that might serve as markers of early stages of thiamine deficits. These include erythrocyte transketolase activity, blood TDP or serum y-glutamyl transferase, which in combination with questionnaire anamnesis, may facilitate early diagnosis to impose easy and efficient treatment with thiamine. 

Herve, C., Beyne, P., Letteron, Ph., and Delacoux E., 1995. Comparison of erythrocyte transketolase activity with thiamine phosphate ester levels in chronic alcoholic patients. Clinica Chimica Acta. 234 91-100. 

Pietrzak, L, and Baczyk, K., 2001. Erythrocyte transketolase activity and guanidine compounds in hemodialysis patients. Kidney International. 59(Suppl. 78) S97-S101. 

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