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aConverted from mg/g creatinine using the factor (x0.376). bBased on a decrease of 25% in red cell thiamine diphosphate (TDP).

aConverted from mg/g creatinine using the factor (x0.376). bBased on a decrease of 25% in red cell thiamine diphosphate (TDP).

popular test, however, is the erythrocyte transketo-lase (ETKL) stimulation test, which measures enzyme activity with and without added TDP. The reference range for ETKL activity in well-nourished, thiamin-adequate people is reported to be 570-830mU/g hemoglobin. The stimulation test measures the proportion of the apoenzyme in red cell homogenate (i.e., the proportion that is not bound to TDP and represents the degree of thiamin deficiency). Studies have shown that results from the urinary assay for thiamin agree reasonably well with those obtained by the ETKL stimulation test.

One of the reasons for the popularity of the ETKL stimulation test is that sensitivity is still good even in the presence of thiamin deficiency. In all other measurements of thiamin status, as deficiency approaches, the quantity of thiamin or its metabolites diminishes in the biological fluid. Low concentrations of a product are usually more difficult to measure and precision deteriorates, or the amount of sample has to be increased to provide sufficient material to detect. In contrast with the ETKL stimulation test, in an acute thiamin deficiency, ETKL activity is maintained and only the amount of TDP decreases, so the test becomes more sensitive. However, in chronic thiamin-deficient states, the apoen-zyme of ETKL is reported to be unstable in vivo, and in the absence of the coenzyme, concentrations of the apoenzyme decrease, with the result that in vitro stimulation may show normal thiamin status. Thus, in situations in which chronic thiamin deficiency is suspected as a result of a long-term marginal thiamin intake, alcohol abuse, or use of diuretics for many months, one or more of the concentration tests may be useful as an adjunct to the stimulation test.

Certain precautions should be taken in handling samples for thiamin analysis. Urine should be acidified to avoid degradation and stored below —20 °C. Heparinized whole blood should be collected and immediately put on ice. For total erythrocyte TDP measurements, cells are separated from plasma within 2h when possible, washed in saline, and diluted 1:1 with saline prior to acidification. Centri-fugation of the acidified mixture provides a clear extract that can be stored for no more than 5 days at 4 °C or longer at <—20 °C. Washed red cells are also used for the ETKL assay. Duplicate tubes of the red cells in saline suspension with and without added TDP are mixed and can be stored at —70 °C prior to enzymatic analysis of ETKL activity. Even at —70 °C, however, storage should be for no more than a few weeks. The ETKL apoenzyme is unstable, and even in the tubes to which TDP has been added, if mixing did not thoroughly expose all apoenzyme to the added coenzyme, deterioration will occur and results will be unreliable.

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Beat The Battle With The Bottle

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