These include pyruvate dehydrogenase (responsible for the conversion of pyruvate to acetyl‐coenzyme A), α‐ketoglutarate dehydrogenase in the Krebs cycle, and the branched chain α‐keto acid dehydrogenase complex. The assay monitors the decrease in absorbance at 340 nm related to NADH oxidation. Holo‐transketolase enzyme concentration is lower in older than younger erythrocytes41 and as holo‐transketolase decreases, there is a consequent increase in the ETKAC.26, 41, The ETKAC assay provides an indirect, functional measure of thiamine status. TEST: 006018 . Between‐individual CV is around 5%. CPT: 86905(x2) Print Share Include LOINC® in print. Agreement in our own laboratory between the two sample types is shown in Figure 2. In this manuscript, we describe the background to the ETK activation assay and discuss aspects relating to reliability and robustly performing, reporting, and interpreting the ETK activity data. A study from 1990 reported within‐individual variation of basal and activated transketolase activity between 3% and 6% measured in 20 samples collected from each of four individuals over 60 days on a constant diet.28. xylulose‐5‐phosphate + ribose‐5‐phosphate ⇆ sedoheptulose‐7‐phosphate + glyceraldehyde‐3‐phosphate, xylulose‐5‐phosphate + erthyose‐4‐phosphate ⇆ fructose‐6‐phosphate + glyceraldehyde‐3‐phosphate, In the current assay, the resultant glyceraldehyde‐3‐phosphate is metabolized to dihydroxyacetone phosphate, which in the final reaction is reduced to glycerol‐3‐phosphate by glycerol‐3‐phosphate dehydrogenase and NADH (Fig. 4). The concentration of total thiamin (free thiamin plus its phosphate esters in whole blood) is 60–120 μg/L, with 90% of the vitamin in erythrocytes and leukocytes. The TPP effect is TKTPP - TK x 100% TKTPP The paired t-test was used toexamine differences designed the assay. We compared erythrocyte TDP with results of the erythrocyte transketolase activation test in 63 patients who were considered at risk of thiamin deficiency. General Information Thiamine pyrophosphate is coenzyme to three systems one of which is transketolase: situated within the hexose monophosphate shunt. 19. The establishment of quality control materials and a quality assurance scheme are recommended to provide reliability. and you may need to create a new Wiley Online Library account. Expected Turnaround Time. The NIHR Cambridge Biomedical Research Centre is a partnership between Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge, funded by the NIHR. ... this test is now considered obsolete. Please check your email for instructions on resetting your password. ETK activity is commonly expressed as a ratio or “activity coefficient” (ETKAC). K.S.J. However, it does not provide information on thiamine status, particularly when status is low, as thiamine may be conserved. Vitamin B1 (thiamine) is an essential nutrient that acts as a cofactor for a number of metabolic processes, particularly in energy metabolism. The erythrocytes … This improved measurement precision, even at observed high absorbance, and the ability to run basal and activated measurements side by side further improved performance.9, 10 For additional convenience and to remove the need for large, expensive equipment, the assay has been further adapted to a 96‐well plate format and measurement with a plate reader.11 The assay as it is currently performed in our laboratory is described herein. 1). After an erythrocyte transketolase test confirmed that my daughter was extremely thiamine deficient, she started taking a form of thiamine that crosses the blood brain barrier. Thiamine diphosphate is a cofactor for transketolase, including erythrocyte transketolase (ETK). Use the link below to share a full-text version of this article with your friends and colleagues. (trans″kēt'ol-ās″) The degree to which transketolase, an enzyme involved in carbohydrate metabolism, rises in response to a loading dose of thiamine pyrophosphate (TPP). After mixing the solution is incubated at 37’ for 15 min and then the reaction rate is measured by recording the absorbance (A) at 339 nm for 15 min. Erythrocyte transketolase levels reliably detect thiamine deficiency but are not necessary for the diagnosis of WE. Erythrocyte thiamine pyrophosphate analysis – This is the most sensitive test. wrote the manuscript. What is Nutritional and Environmental Medicine? observed greater deterioration of the ETKAC after storage of washed erythrocytes at −20 °C in participants in an insufficient group (ETKAC > 1.15) group than in a sufficient group (ETKAC < 1.15), and they speculate this may be due to the stability of different isoenzymes. Reagents required for the ETKAC assay. In our tests for vitamins B1 we measure the change in enzyme activity (erythrocyte transketolase[ETK]) before and after the addition of excess vitamin. Thiamine status can be assessed by three types of tests: determination of erythrocyte transketolase activity, the urinary excretion of thiamine before and after thiamine administration, and serum, erythrocyte, or whole blood thiamine levels. In our laboratory, whole blood samples from 15 participants kept at 4 °C were also observed to be stable for 24 h, with no significant change in the ETKAC observed in LH‐treated whole blood (% geometric mean change (95% confidence interval): −0.3 (−2 to 2)) or in EDTA‐treated whole blood (−1 (−3 to 1)), and they showed good agreement (Fig. However, the methodology to measure ETK activity has received less attention. It is also an enzyme classified as a(n) transketolase. The impact of clinically observed thiamine deficiency, ThDP concentration, their effect on transketolase enzyme, and consequences for interpretation of transketolase activity have been a topic of discussion for many years.17, 38 It has been suggested that liver disease may directly affect apo‐transketolase concentration, causing apparently normal ETKAC in the presence of clinical symptoms of thiamine deficiency. The ETK activity assay in its early form for the application of thiamine deficiency assessment was established by Brin7 and was later improved and adapted as understanding and technology progressed. Traditionally the erythrocyte transketolase saturation test, which is a measure of the stimulation of the transketolase reaction, has been used to assess thiamin status. The activation of red blood cell transketolase in groups of patients especially at risk from thiamin deficiency - Volume 17 Issue 2 - Marina D. Jeyasingham, Oliver E. Pratt, Alistair Burns, George K. Shaw, Allan D. Thomson, Andrew Marsh A potassium dichromate solution of about 0.4 g/l is used as Enzyme activities reportedly remain stable at −70 °C for over 1 year.2 Storage of washed erythrocytes and hemolysates at −70 °C or below is recommended.16. Clin Chem. The assay monitors the rate of oxidation of NADH by measuring the decrease in absorbance at 340 nanometers. Large increases in enzyme activity after TPP loading indicate the tested patient has a thiamine deficiency. The ETK activity assay as an indirect, functional marker of thiamine status has been used for over 50 years. Harmonization of the protocol will provide the basis for the development of internationally recognized cutoffs for thiamine insufficiency. Test number copied. Two measurements are made to determine the EKTAC: (1) the basal activity of ETK and (2) “activated” or “stimulated” ETK activity by the addition of exogenous excess ThDP. TRANSKETOLASE IN ERYTHROCYTES 381 0.01 ml GDH-TIM and 0.05 ml NADH. Thiamine measurement in patients with behavioral changes, eye signs, gait disturbances, delirium, and encephalopathy; or in patients with questionable nutritional status, especially those who appear at risk and who also are being given insulin for hyperglycemia Urinary thiamine excretion may correlate with all levels of thiamine intake but low intakes. In order to investigate the potential impact of subclinical deficiency and to improve our understanding of the global prevalence of thiamine deficiency, biomarkers to assess thiamine status are essential. While a venous blood sample is a common sample type, collection and processing of blood samples in the field or limited‐resource settings present a number of challenges, including laboratory infrastructure for washing of erythrocytes and cold storage. Medical Dictionary, © 2009 Farlex and Partners The use of whole blood, a more convenient sample compared with washed erythrocytes, has also been considered. 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. Washed erythrocytes are the recommended sample type. Any queries (other than missing content) should be directed to the corresponding author for the article. Slope (95% CI) = 1.06 (0.92–1.20); intercept (95% CI) = −0.07 (−0.24 to 0.09) (, © 2021 The New York Academy of Sciences. described a DBS method for ThDP that showed a high degree of correlation with whole blood results (r = 0.964; P < 0.0001), although ThDP in DBSs was not stable at room temperature.37 Further work is required to determine the feasibility of measuring ETKAC in whole blood as either a venous sample or a DBS. Test Resources. Reference ranges: B1 (Thiamine) 1.15 normal, 1.15 - 1.25 borderline, >1.25 deficient Results expressed as ratio of activated to basal activity in IU/gHb. Erythrocyte Sedimentation Rate. Authors J E Rossouw, D Labadarios, N Krasner, M Davis, R Williams. 3222680. Reference ranges:B1 (Thiamine) <1.15 normal, 1.15 - 1.25 borderline, >1.25 deficientResults expressed as ratio of activated to basal activity in IU/gHb, B vitamin profile and full Vitamin Profile. For further details please contact the laboratory at: lab@xxxxbiolab.co.uk. The difficulty of obtaining blood samples in sufficient volume from thiamine‐deficient individuals results in a lack of quality control materials that include the thiamine‐deficient range. Recently, Huang et al. Doctors & Nutritionists Available for Consultation. The roles of thiamin and its phosphate esters as a coenzyme in carbohydrate metabolism and in the process of nerve conduction are now well established (1)(2). In our tests for vitamins B1 we measure the change in enzyme activity (erythrocyte transketolase[ETK]) before and after the addition of excess vitamin. Online Version of Record before inclusion in an issue. ThDP is an essential cofactor for a number of metabolic enzymes, particularly those associated with oxidative and nonoxidative carbohydrate metabolism. reviewed and approved the manuscript and accept responsibility for the contents. What is Nutritional and Environmental Medicine? 2000 May;46(5):704-710. Part III: the apoenzyme stimulation tests for vitamin B1, B2 and B6 adapted to the Cobas‐Bio analyzer, National Diet and Nutrition Survey Results from Years 1, 2, 3 and 4 (combined) of the Rolling Programme (2008/2009–2011/2012), The relationship between the thiamin pyrophosphate effect and the saturation status of the transketolase with its coenzyme in human erythrocytes, EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B, Thiamin and riboflavin status of Taiwanese elementary schoolchildren, Erythrocyte transketolase activity, markers of cardiac dysfunction and the diagnosis of infantile beriberi, Comparison of thiamin diphosphate high‐performance liquid chromatography and erythrocyte transketolase assays for evaluating thiamin status in malaria patients without beriberi, Kinetic transketolase assay: use of whole‐blood hemolysate as the sample, Leukocyte transketolase activity as an indicator of thiamin nutriture in rats, Leukocyte transketolase activity: an indicator of thiamin nutriture, A comparison of transketolase assay and transketolase and lactate dehydrogenase activity levels in whole blood and red cell hemolysates and in leukocytes, A fluorimetric method for measurement of erythrocyte transketolase activity, Changes in erythrocyte transketolase activity and the thiamine pyrophosphate effect during storage of blood, Evaluation of methods of coenzyme activation of erythrocyte enzymes for detection of deficiency of vitamins B1, B2, and B6, Variability in man of the levels of some indices of nutritional status over a 60‐d period on a constant diet, National Diet and Nutrition Survey. However, the test is somewhat nonspecific, as other factors may decrease transketolase activity. If you do not receive an email within 10 minutes, your email address may not be registered, kerry.jones@mrc-epid.cam.ac.uk, MRC Elsie Widdowson Laboratory, Cambridge, UK. Application: Rarely indicated for the detection of vitamin B 1 deficiency. Mushrooms; Lettuce, spinach and green peas; Cereals, rye, and wheat germ; Eggplants, sprouts, tomatoes; Potatoes and kidney beans; Alcohol and Low Thiamine Puxty et al. The Test of Choice for Assessing Thiamine Deficiency Erythrocyte is the technical name for red cells. This will ensure that the ETK activity assay remains an important method for the assessment of thiamine status. All Rights Reserved, Annals of the New York Academy of Sciences, Transactions of the New York Academy of Sciences, orcid.org/https://orcid.org/0000-0002-7380-9797, orcid.org/https://orcid.org/0000-0001-9998-051X, I have read and accept the Wiley Online Library Terms and Conditions of Use, Thiamine deficiency disorders: diagnosis, prevalence, and a roadmap for global control programs, Thiamine deficiency in tropical pediatrics: new insights into a neglected but vital metabolic challenge, Comparable performance characteristics of plasma thiamine and erythrocyte thiamine diphosphate in response to thiamine fortification in rural Cambodian women, Vitamin B1 in critically ill patients: needs and challenges, Effects of thiamine deficiency and of oxythiamine on rat tissue transketolase, A NADH‐dependent transketolase assay in erythrocyte hemolysates, Adaptation of coenzyme stimulation assays for the nutritional assessment of vitamins B1, B2 and B6 using the Cobas Bio centrifugal analyser, Clinical chemical methods for the routine assessment of the vitamin status in human populations. The spectrophotometric method in the modification by Bayoumi and Rosalki8and Smeets et al9was used to measure the transketolase activity in erythrocytes (TK) being expressed as units per gram of hemoglobin. are supported by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre (IS‐BRC‐1215‐20014). Plate map for the ETKAC analysis. 2 isoforms have been identified. In this paper, we provide a detailed protocol for the measurement of ETK activity that can be used as a foundation for assay harmonization. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. While transketolase activity was higher in whole blood, the transketolase activity coefficient was similar between whole blood and erythrocyte hemolysates.20, 23 However, there are concerns around the use of whole blood, partly due to between‐person variability in leukocyte count, particularly in clinical conditions that may impact leukocyte number.24. Price et al. File S1. SUMMARY. Indirect: spectrophotometric assay of red cell transketolase with and without added thiamin pyrophosphate. In thiamine sufficiency, addition of exogenous ThDP will make little difference to the enzyme activity and hence an ETKAC close to 1 will be obtained. Takeuchi et al. The populations most at risk are breastfed infants of thiamine‐deficient mothers in low‐income countries, particularly those with poor diet diversity and where rice is the staple food.2, 3 In high‐income countries, deficiency is rare but may be present in the elderly, chronic alcoholics, and persons with acute or chronic medical conditions associated with malnutrition.1 In addition to the effect of acute deficiency, evidence also exists that subclinical deficiency may have long‐term effects on cognition and gross motor skills.2. Furthermore, ETKAC provides a potentially better marker of long‐term thiamine status that is less affected than ThDP by acute changes in thiamine intake.43. It mediates transfer of CH2OHC=O between donor and acceptor sugars. The views expressed are those of the authors and not necessarily those of the NHS, NIHR, or Department of Health and Social Care. An erythrocyte transketolase isoenzyme pattern associated with the Wernicke-Korsakoff syndrome. Testing may be indicated in suspected alcoholic or nutritional cardiomyopathy or neuropathy and in Wernicke-Korsakoff syndrome. Direct measurements of the vitamins are affected by dietary intake and nutritional supplementation. Capillary blood sampling and dried blood spots (DBSs) provide a more convenient sample. This test may also be used to help diagnose and/or monitor a number of diseases that affect the production or lifespan of red blood cells. Transketolase activity was measured in the erythrocyte haemolysates of 14 patients with chronic uraemia and in 16 healthy controls in the presence of TPP and following TPP saturation. Working off-campus? Automated Modified Westergren Photometrical capillary stopped flow kinetic analysis. ETKAC data from the UK National Diet and Nutrition Survey (NDNS) Rolling Programme (Years 7–8 (2014–2016))29 are presented in Table 1 and show the range of values in a largely thiamine‐replete population. Slope (95% CI) = 0.88 (0.37–1.39); intercept (95% CI) = 0.26 (−0.44 to 0.68). A venous whole blood sample is required for the determination of ETKAC (and whole blood ThDP). Patients received either intravenous Pabrinex (thiamine) supplemented with magnesium sulphate (n = 18) or Pabrinex only (n = 18). K.S.J., D.A.P., L.J.C., and A.K. Test Details. In the erythrocyte transketolase activity assay, the … The establishment of a network of international laboratories willing to share samples for an interlaboratory comparison could provide a first step in the development of an external quality assurance scheme. The diagnosis of beriberi is assisted by a dietary history suggestive of a low thiamine intake and clinical manifestations. However, objective biochemical tests of thiamine status, particularly measurement of erythrocyte transketolase activity (ETKA) and the thiamine pyrophosphate effect (TPPE), provide a sensitive test for thiamine deficiency where there are laboratory facilities … Body levels of B vitamin levels are best determined using enzyme dependent activity tests. As an index of thiamine intake and clinical manifestations the development of internationally recognized for! For Assessing thiamine deficiency are recognized as beriberi, although clinical symptoms are nonspecific and of... After storage for 6 months at −70 °C number of metabolic enzymes, particularly those associated oxidative! Automated Modified Westergren Photometrical capillary stopped flow kinetic analysis marker of thiamine.. Capillary stopped flow kinetic analysis in erythrocyte hemolysates by a dietary history suggestive of a low thiamine intake nutritional. 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