Erythrocyte transketolase activity coefficient (ETKAC) assay protocol for the assessment of thiamine status

Abstract

Vitamin B1 (thiamine) is an essential nutrient that acts as a cofactor for a number of metabolic processes, particularly in energy metabolism. Symptoms of classic thiamine deficiency are recognized as beriberi, although clinical symptoms are nonspecific and recognition of subclinical deficiency is difficult. Therefore, reliable biomarkers of thiamine status are required. Thiamine diphosphate is a cofactor for transketolase, including erythrocyte transketolase (ETK). The ETK activity assay as an indirect, functional marker of thiamine status has been used for over 50 years. The ETK activity assay provides a sensitive and specific biomarker of thiamine status; however, there is a lack of consensus over the cutoffs for deficiency, partly due to a lack of assay harmonization. Here, we provide a step-by-step protocol for the measurement of ETK activity and the calculation of the ETK activity coefficient, including detailed explanations of equipment and chemicals required and guidance for quality control procedures. Harmonization of the protocol will provide the basis for the development of internationally recognized cutoffs for thiamine insufficiency. The establishment of quality control materials and a quality assurance scheme are recommended to provide reliability. This will ensure that the ETK activity assay remains an important method for the assessment of thiamine status.

Keywords: ETKAC; beriberi; thiamine; vitamin B1.

Figure 1

The enzymatic reactions in the ETK activity assay. The assay monitors the decrease in absorbance at 340 nm related to NADH oxidation.

Figure 2

Deming regression of ETKAC measured in erythrocyte hemolysates collected in either ETDA or lithium heparin (LH) blood tubes from 15 adults. Open circles, observed data points; solid line, regression line; dashed line, line of equality. Slope (95% CI) = 0.88 (0.37–1.39); intercept (95% CI) = 0.26 (−0.44 to 0.68).

Figure 3

Deming regression of ETKAC measured in erythrocyte hemolysates prepared from LH and EDTA whole blood collected from 15 adults and processed within 2 h of collection or after refrigeration for 24 hours. Dashed line is the line of equality.

Figure 4

Deming regression of repeat analysis of erythrocyte hemolysates after storage for 6 months at −70 °C. Open circles, observed data points; solid line, regression line; dashed line, line of equality. Slope (95% CI) = 1.06 (0.92–1.20); intercept (95% CI) = −0.07 (−0.24 to 0.09) (n = 59).