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.

Delayed Processing of Chilled Whole Blood for 24 Hours Does Not Affect the Concentration of the Majority of Micronutrient Status Biomarkers

“…After removal of plasma, washed erythrocytes were prepared by adding an approximate volume of normal saline to equal the original whole-blood volume in the tube, gentle inversion, and centrifugation at 2000 × g at 4°C for 10 minutes. The supernatant was removed and the process was repeated twice more ( 20 ). Washed erythrocytes were stored at −70°C until analysis.…”

Hiding in Plain Sight: Modern Thiamine Deficiency

“…Values >25% are considered severe and those >40% are noted with WE. [15,16] Experimentally, however, the definition of deficiency varies based upon the purpose of the study. Comparison of lab tests, for example, considers activation >25% deficient [17].…”

A fresh look at thiamine deficiency—new analyses by the global thiamine alliance

“…Harmonization of protocols is an important step toward ensuring the comparability of studies performed in different laboratory settings and over time. 14 Populations at risk Thiamine deficiency has typically been associated with a reliance on poor diets in low-and middle-income countries (LMICs) or with alcoholism in adults (mostly manifested as Wernicke's encephalopathy). However, as Gomes et al demonstrate in their literature review, adult thiamine deficiency is not exclusive to LMICs or alcoholic patients in high-income settings.…”

“…Harmonization of protocols is an important step toward ensuring the comparability of studies performed in different laboratory settings and over time. 14 …”

“…present a step‐by‐step protocol for the measurement of erythrocyte transketolase (ETK) activity and calculation of the ETK activity coefficient, including a description of equipment and chemicals required, as well as guidance for quality control procedures. Harmonization of protocols is an important step toward ensuring the comparability of studies performed in different laboratory settings and over time 14 …”