ASSAY FOR THIAMINE DEFICIENCY:

One method for evaluation of thiamine status is the measurement of erythrocyte (red blood cell) transketolase activity. Red blood cells, which lack mitochondria, have no alternative means of generating NADPH save the pentose phosphate pathway. Also, NADPH is required to reduce glutathione in order to maintain the normal structure of red blood cell and maintain hemoglobin in the ferrous state.  Therefore, the pentose phosphate pathway is essential in red blood cells.  Transketolase is a thiamine pyrophosphate-requiring enzyme, which catalyzes reactions in the pentose phosphate pathway.  So the level of transketolase activity in the red blood cell is a reliable diagnostic indicator of thiamine status.

The erythrocyte transketolase test requires a sample of hemolyzed blood to be incubated with excess ribose 5-phosphate (or xylulose 5-phosphate), in the presence of excess added thiamine pyrophosphate (matched with a control that has no added TPP).  After the incubation period, one then measures the amount of substrate remaining and the amount of product formed.  These concentrations are measured by using high performance liquid chromatography (HPLC), and a UV absorbance detector.  The effluent is then monitored by the UV-detector. Any enhancement in enzyme activity resulting from the added thiamine pyrophosphate indicates that the sample was originally deficient in thiamine to some extent. The extent of deficiency in thiamine is expressed in percent stimulation over the control value (see table below). 

Classification                                                            TPP stimulation

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