Comparison of Thiamin Diphosphate High-Performance Liquid Chromatography and Erythrocyte Transketolase Assays for Evaluating Thiamin Status in Malaria Patients without Beriberi

Abstract

Thiamin deficiency, or beriberi, is an increasingly re-recognized cause of morbidity and mortality in the developing world. Thiamin status has traditionally been measured through the erythrocyte activation assay (ETKA) or basal transketolase activity (ETK), which indirectly measures thiamin diphosphate (TDP). Thiamin diphosphate can also be measured directly by high-performance liquid chromatography (HPLC), which may allow a more precise estimation of thiamin status. We compared the direct measurement of TDP by HPLC with basal ETK activity and ETKA in 230 patients with Plasmodium falciparum malaria in rural southern Laos but without overt clinical beriberi, as part of a trial of thiamin supplementation. Admission thiamin status measured by basal ETK activity and ETKA (α) was compared with thiamin status assessed by the measurement of TDP by HPLC; 55% of 230 included patients were male, and the median age was 10 (range 0.5-73) years. Using α ³ 25% as the gold standard of thiamin deficiency, the sensitivity of TDP < 275 ng/gHb as a measure of thiamin deficiency was 68.5% (95% CI: 54.4-80.5%), with specificity of 60.8 (95% CI: 53.2-68.1%). There was a significant inverse correlation between the results of the two tests (Kendall's tau = −0.212, P < 0.001). Basal ETK activity was also significantly positively correlated with TDP levels (Kendall's tau = 0.576, P < 0.001). Thiamin diphosphate measurement may have a role in measuring thiamin levels in clinical settings. Further studies evaluating TDP concentration in erythrocytes with basal ETK activity and ETKA (α) in beriberi patients would help establish comparative values of these assays.

Protocol and application of basal erythrocyte transketolase activity to improve assessment of thiamine status

“…Basal ETK activity is reported to be more closely related to ThDP than ETKac, 18 possibly because of the potential ambiguity in activity coefficients when apoenzyme levels are low, and was observed to better predict infantile beriberi than ETKac. 17 Other studies have reported that ETKac provides a more robust and sensitive marker of thiamine status.…”

“…Mothers in Laos had a mean basal ETK activity of 0.58 U/gHb. 17 A number of other studies performed in Laos reported values in malaria-infected patients 18,19 and in infants with 17 and without 17,44 clinical signs of thiamine deficiency.…”

“…This was in contrast to the use of ETKac cut-offs that showed no discrimination between cases and controls. 17 The cut-off value of 0.59 has since been used in other studies, 18,23,35 but remains unsubstantiated. Soukaloun et al 17 also reported reference ranges of between 0.47 and 0.57 U/gHb for children and women aged between 4 and 49 years and derived from UK NDNS data.…”

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

“… 18 A recent study of children and adults found that while both basal and ETKAC correlated with erythrocyte ThDP, the correlation was stronger for basal activity. 19 Basal (or activated ETK) activity needs to be expressed per mass unit of hemoglobin, and it is necessary to measure the hemoglobin concentration in the same lysate as used for the ETK activity assay. In addition, it is necessary to calculate the factor needed to convert rate to activity.…”

“…However, the use of basal ETK activity rather than ETKAC has also been recommended, particularly for the assessment of thiamine status in infants due to potential low levels of apoenzyme caused by long‐term low exposure to thiamine 18 . A recent study of children and adults found that while both basal and ETKAC correlated with erythrocyte ThDP, the correlation was stronger for basal activity 19 . Basal (or activated ETK) activity needs to be expressed per mass unit of hemoglobin, and it is necessary to measure the hemoglobin concentration in the same lysate as used for the ETK activity assay.…”