Thiamin transketolase activation

Ketols can also be formed enzymatically by cleavage of an aldehyde (step a, Fig. 14-3) followed by condensation with a second aldehyde (step c, in reverse). An enzyme utilizing these steps is transketolase (Eq. 17-15),132b which is essential in the pentose phosphate pathways of metabolism and in photosynthesis. a-Diketones can be cleaved (step d) to a carboxylic acid plus active aldehyde, which can react either via a or c in reverse. These and other combinations of steps are often observed as side reactions of such enzymes as pyruvate decarboxylase. A related thiamin-dependent reaction is that of pyruvate and acetyl-CoA to give the a-diketone, diacetyl, CH3COCOCH3.133 The reaction can be viewed as a displacement of the CoA anion from acetyl-CoA by attack of thiamin-bound active acetaldehyde derived from pyruvate (reverse of step d, Fig. 14-3 with release of CoA). 

Early studies showed that the development of neurological abnormalities in thiamin deficiency did not follow the same time course as the impairment of pyruvate and 2-oxoglutarate dehydrogenase or transketolase activities. The brain regions in which metabolic disturbances are most marked were not those that are vulnerable to anatomical lesions. These studies suggested a function for thiamin in the nervous system other than its coenzyme role. 

On the basis of depletion/repletion studies, an intake ofO.2 mgper 1,000kcal is required to maintain normal urinary excretion, but an intake of 0.3 mg per 1,000 kcal is required for a normal transketolase activation coefficient. At low levels of energy intake, there will be a requirement for metabolism of endogenous substrates and to maintain nervous system thiamin triphosphate. 

Measurement of red ceil transketolase activity can be used as an index of thiamine deficiency. 

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-... 

The transketolase activation test is in reality two tests one a measurement of basal activity and the other the degree to which the basal activity can be increased by exogenous thiamine pyrophosphate, and each may be influenced by different factors. There is evidence that chronic deficiency states of thiamine may down regulate synthesis of the apoen-zyme. In comparison studies against erythrocyte TPP concentrations, better correlations were obtained with basal activity rather than the activation coefficient. ... 

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. 

B, (Thiamin) Beri-Beri Plasma levels or RBC transketolase activation... 

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 . 

Beriberi is associated with low transketolase activity in the blood, and determination of transketolase activity may be a helpful diagnostic tool in detecting mild forms of thiamine deficiencies. The fact that adding thiamine pyrophosphate to the system restores transketolase activity confers specificity to this procedure. Although the decrease in transketolase activity is easy to explain in view of the role that thiamine plays in that reaction, the mechanism of the alteration in adenosine-5 -phosphatase is not so obvious. The total activity is decreased despite a slight increase of adenosine phosphatase in the nuclei, and the decrease in total activity results from a marked decrease of adenosine phosphatase in the fibers. Increased thiamine pyrophosphate and alkaline phosphatase have also been observed in the optic tectum of the deficient chicken, but these changes remain unexplained. 

Zieve, L., Doizaki, W. M., and Stenroos, L. E., 1968a, Effect of magnesium deficiency on blood and liver transketolase activity and on the recovery of enzyme activity in thiamine-deficient rats receiving thiamine, J. Lab. Clin. Med. 72 268. 

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

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. 

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. 

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. 

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