In addition, we present a step‐by‐step protocol (see File S1, online only) that provides detailed guidance and should enable laboratories to set up the method independently and improve harmonization. In whole blood kept at 4 °C or at room temperature, the ETKAC was stable for up to 24 h, decreasing thereafter;25, 26 however, basal activity was reportedly stable for up to 4 days.25. Enter your email address below and we will send you your username, If the address matches an existing account you will receive an email with instructions to retrieve your username. has an advisory role at the NIHR BRC Nutritional Biomarker Laboratory. A small number of studies have investigated the stability of ETK activity and ETKAC across a number of analytical stages, including in whole blood and hemolysates, and at a range of temperatures from refrigeration at 4 °C to frozen storage at −20 and −70 °C, possibly reflecting the availability of cold storage in different clinical and field site settings. Reference Interval: Consult pathologist. The diagnosis of beriberi is assisted by a dietary history suggestive of a low thiamine intake and clinical manifestations. 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. Reagents required for the ETKAC assay. Results: The method was linear to at least 200 microgram/L. Thiamine diphosphate is a cofactor for transketolase, including erythrocyte transketolase (ETK). The authors declare no competing interests. The advantages and disadvantages of both the direct and indirect assays were reviewed recently,1, 2, 6 including a detailed description of the direct measurement of ThDP. Application: Rarely indicated for the detection of vitamin B 1 deficiency. Addition of exogenous ThDP activates apo‐transketolase and the stimulated activity represents the activity of both the (now‐activated) apo‐transketolase and holo‐transketolase. METHODS: Baseline erythrocyte transketolase activities were measured on blood samples collected from 36 chronic alcoholic patients presenting acutely to the Accident and Emergency department. Medical Dictionary, © 2009 Farlex and Partners 2 isoforms have been identified. wrote the manuscript. Talwar D, Davidson H, Cooney J, St JO'Reilly D. Vitamin B(1) status assessed by direct measurement of thiamin pyrophosphate in erythrocytes or whole blood by HPLC: comparison with erythrocyte transketolase activation assay. 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. Traditionally the erythrocyte transketolase saturation test, which is a measure of the stimulation of the transketolase reaction, has been used to assess thiamin status. 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. Large increases in enzyme activity after TPP loading indicate the tested patient has a thiamine deficiency. The transketolase method is an indirect assessment. A stimulation of greater than 16% has been taken as a thiamin deficiency. Green (lithium heparin)- must reach the lab within 24 hours of collection. 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 erythrocytes … In the erythrocyte transketolase activity assay, the … designed the assay. Assays moved from the less specific colorimetric determination of substrate disappearance or appearance of end products to UV detection of NADH.8 The assay relies on the detection of small changes in enzyme activity and early methods lacked the necessary sensitivity and had relatively poor precision. Some of the experimental work was performed at the MRC Elsie Widdowson Laboratory, Cambridge, UK, which closed in December 2018. We compared erythrocyte TDP with results of the erythrocyte transketolase activation test in 63 patients who were considered at risk of thiamin deficiency. TEST: 006018 . Test Details. Mushrooms; Lettuce, spinach and green peas; Cereals, rye, and wheat germ; Eggplants, sprouts, tomatoes; Potatoes and kidney beans; Alcohol and Low Thiamine 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. (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). Erythrocyte transketolase levels reliably detect thiamine deficiency but are not necessary for the diagnosis of WE. 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. In thiamine insufficiency or deficiency, the addition of exogenous ThDP has a progressively greater effect on ETKAC, which provides a continuum of thiamine status.13, Although there is no international consensus on cutoffs, the commonly used threshold for risk of deficiency is an ETKAC of >1.25.13, 14 An ETKAC of <1.15 indicates sufficiency and values between 1.15 and 1.25 suggest a low risk of clinical deficiency.13 Others have suggested values of ≥1.2 indicate deficiency.15 Beriberi is typically associated with ETKAC values >1.4.2, The use of ETKAC rather than absolute measures of ETK activity is preferred for three main reasons: (1) the between‐subject variation in basal activity is large; (2) it is assumed, but not certain, that apoenzyme levels are not affected by vitamin deficiencies;5 and (3) it reduces the need for the precise definition of assay conditions (e.g., optical path length), which are critical to calculate absolute enzyme activities.16 Others have suggested using combinations of the stimulated ETK activity and the ETKAC in interpretation.17. Therefore, there is a need to develop quality control and reference materials with consensus values spanning both sufficiency and deficiency, which can be supplied to laboratories in order to monitor, understand, and standardize assay performance. This will ensure that the ETK activity assay remains an important method for the assessment of thiamine status. ETKAC has advantages over methods for the direct measurement of ThDP, including relative ease and the requirement for less specialized equipment, making the assay potentially more affordable and sustainable. Dashed line is the line of equality. performed the experiments. 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. The views expressed are those of the authors and not necessarily those of the NHS, NIHR, or Department of Health and Social Care. Targets of <5% are recommended. The ThDP‐dependent enzyme ETK catalyzes the metabolism of pentoses in two reactions in the pentose phosphate pathway: The enzymatic reactions in the ETK activity assay. Patients received either intravenous Pabrinex (thiamine) supplemented with magnesium sulphate (n = 18) or Pabrinex only (n = 18). Provided below are ELISA kits targeting transketolase, a human protein encoded by TKT. Washed erythrocytes are the recommended sample type. 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. A venous whole blood sample is required for the determination of ETKAC (and whole blood ThDP). Nixon PF, Kaczmarek MJ, Tate J, Kerr RA, Price J. K.S.J., D.A.P., L.J.C., and A.K. are supported by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre (IS‐BRC‐1215‐20014). and you may need to create a new Wiley Online Library account. 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. In addition, it is necessary to calculate the factor needed to convert rate to activity. 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 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. What is Nutritional and Environmental Medicine? Puxty et al. Methodology. Authors J E Rossouw, D Labadarios, N Krasner, M Davis, R Williams. Please check your email for instructions on resetting your password. 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. 3). 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. 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. In the first reaction of the non-oxidative pentose phosphate pathway, the cofactor thiamine diphosphate accepts a 2-carbon fragment … 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 method for measuring erythrocyte transketolase activity (ETKA) is described that is precise, economical of reagents and capable of high throughput with partial or full automation. This enzyme is important in erythrocyte metabolism and its activity can therefore be used as an index of thiamine nutrition. 10794754 LabCorp test details for Red Blood Cell (RBC) Antigen Typing: E/e ... Red Blood Cell (RBC) Antigen Typing: E/e. 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. In conjunction with whole blood or erythrocyte transketolase activity preloading and postloading, a thiamine loading test is the best indicator of thiamine deficiency. Online Version of Record before inclusion in an issue. ETK activity is commonly expressed as a ratio or “activity coefficient” (ETKAC). All rights reserved. In our tests for vitamins B1 we measure the change in enzyme activity (erythrocyte transketolase[ETK]) before and after the addition of excess vitamin. Between‐individual CV is around 5%. Genetic differences leading to different transketolase isoforms that may have different activities or stabilities30 or be associated with the manifestation of thiamine deficiency in Wernicke–Korsakoff syndrome have been suggested.30, 31 However, other studies have found no evidence for isoforms or disease associations and suggest that earlier observations were due to the methodologies used.32-36. 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. Test Resources. Recently, Huang et al. However, it does not provide information on thiamine status, particularly when status is low, as thiamine may be conserved. The measurement of erythrocyte transketolase activity enables evaluation of the thiamine status and therapeutic decisions in the disorders … The assay monitors the rate of oxidation of NADH by measuring the decrease in absorbance at 340 nanometers. 19. The ETK activity assay as an indirect, functional marker of thiamine status has been used for over 50 years. 1). demonstrated in experiments with apo‐transketolase enzymes that the ETKAC reflects the degree of saturation of ETK with ThDP.12. Open circles, observed data points; solid line, regression line; dashed line, line of equality. The results of the basal and stimulated activities are commonly presented as a ratio or activity coefficient, the ETKAC calculated as (stimulated activity)/(basal activity). If you do not receive an email within 10 minutes, your email address may not be registered, 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. The assay was further refined and adapted for semiautomated clinical chemistry analyzers. However, other studies posit that the decrease in apo‐transketolase is a response to thiamine deficiency.17, 38 Either way, chronic thiamine deficiency may cause loss of apo‐transketolase and evidence suggests that this is caused by an effect on synthesis rather than catabolism of transketolase.39 The impact of this effect on interpretation of the EKTAC is, however, uncertain. B1 vitamin deficiency was studied by the erythrocyte enzyme activation test in patients with chronic uraemia. A potassium dichromate solution of about 0.4 g/l is used as Takeuchi et al. For Rh phenotyping, refer to test 058008. L.J.C. There is also a need to develop an external quality assurance scheme to provide for the independent monitoring and assessment of laboratory performance. Erythrocyte transketolase – This is a very sensitive test. Urinary thiamine excretion may correlate with all levels of thiamine intake but low intakes. Figure P1. The basal test represents the active holo‐transketolase activity. Expected Turnaround Time. Clin Chem. 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. Direct measurements of the vitamins are affected by dietary intake and nutritional supplementation. 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. 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. Transketolase encoded by the TKT gene is an enzyme of both the pentose phosphate pathway in all organisms and the Calvin cycle of photosynthesis.It catalyzes two important reactions, which operate in opposite directions in these two pathways. 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. The ratio of activated to basal activity gives the ETKAC and provides a proxy measure for the in vivo activity of ETK and an indication of the availability of ThDP and thiamine status. 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. Any queries (other than missing content) should be directed to the corresponding author for the article. 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. Indirect: spectrophotometric assay of red cell transketolase with and without added thiamin pyrophosphate. However, the test is somewhat nonspecific, as other factors may decrease transketolase activity. Slope (95% CI) = 0.88 (0.37–1.39); intercept (95% CI) = 0.26 (−0.44 to 0.68). Clinical conditions, for example, certain anemias or diseases that affect erythrocyte cell survival time and the age distribution, may also affect the interpretation of ETKAC. Erythrocyte transketolase (TK; EC 2.2.1.1), a thiamine-dependent enzyme, is widely used ... the test is repeated with the addition of 10 J.lL of TPP solution prior to incubation. Included in Profiles: The most common test for this condition is the erythrocyte transketolase assay, which directly measures thiamine pyrophosphate in the red blood cells from a blood sample. 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. 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. Deming regression of repeat analysis of erythrocyte hemolysates after storage for 6 months at −70 °C. Symptoms of classic thiamine deficiency are recognized as beriberi, although clinical symptoms are nonspecific and recognition of subclinical deficiency is difficult. Improvements in harmonization will also provide opportunity for investigating outstanding questions related to thiamine physiology and EKTAC assays, including the establishment of cutoffs for deficiency, characterizing agreement with other markers of thiamine status (e.g., ThDP), and to better understand conditions where interpretation of ETKAC may be confounded. Since transketolase activity requires thiamin, decreased transketolase activity was higher in whole blood and erythrocyte hemolysates lab 24. 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But are not necessary for the determination of ETKAC measured in erythrocyte hemolysates storage... Control materials and a quality assurance scheme to provide reliability thiamin, transketolase!