Subject transketolase

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%. 

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 . 

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). 

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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