In humans, the majority of thiamine exists in phosphorylated forms: thiamine monophosphate, thiamine diphosphate (ThDP) (also known as thiamine pyrophosphate), and thiamine triphosphate. 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. 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. Authors J E Rossouw, D Labadarios, N Krasner, M Davis, R Williams. L.J.C. 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. Thiamine (vitamin B1) is an essential micronutrient. Table P1. The between-run CV was <8%. None found for this test Please visit our Clinical Education Center to stay informed on any future publications, webinars, or other education opportunities. The assay monitors the decrease in absorbance at 340 nm related to NADH oxidation. There is also a need to develop an external quality assurance scheme to provide for the independent monitoring and assessment of laboratory performance. 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. Working off-campus? The assay monitors the rate of oxidation of NADH by measuring the decrease in absorbance at 340 nanometers. 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. 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. It mediates transfer of CH2OHC=O between donor and acceptor sugars. CPT: 86905(x2) Print Share Include LOINC® in print. The establishment of quality control materials and a quality assurance scheme are recommended to provide reliability. 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. Plate map for the ETKAC analysis. 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. Large increases in enzyme activity after TPP loading indicate the tested patient has a thiamine deficiency. What is Nutritional and Environmental Medicine? This protein is 623 amino acids long, with a mass of 67.9 kDa, and is a member of the Transketolase family. Leukocytes have relatively large amounts of thiamine, and transketolase activity can, therefore, be considerably higher in whole blood compared with erythrocytes.8, 20 The use of leukocytes21, 22 to assess transketolase activity has been reported but has not been applied routinely, possibly because the shorter half‐life of leukocytes compared with erythrocytes would result in a biomarker of thiamine status that is only short term. A red blood cell (RBC) count is typically ordered as part of a complete blood count (CBC) and may be used as part of a health checkup to screen for a variety of conditions. In the first reaction of the non-oxidative pentose phosphate pathway, the cofactor thiamine diphosphate accepts a 2-carbon fragment … An erythrocyte transketolase isoenzyme pattern associated with the Wernicke-Korsakoff syndrome. ETK activity is commonly expressed as a ratio or “activity coefficient” (ETKAC). wrote the manuscript. Clin Chem. Protocols for (1) processing of whole blood samples required to produce washed erythrocyte specimens for the erythrocyte transketolase activity coefficient (ETKAC) assay, and (2) the measurement of the erythrocyte transketolase activity coefficient (ETKAC). 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. 2 isoforms have been identified. 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 … 2000 May;46(5):704-710. 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. While transketolase activity was higher in whole blood, the transketolase activity coefficient was similar between whole blood and erythrocyte hemolysates. Reagents required for the ETKAC assay. ThDP is the major form, making up around 80% of total blood thiamine and is mainly present in erythrocytes and white blood cells. TEST: 006018 . Results from Years 7 and 8 (combined) of the Rolling Programme (2014/15 to 2015/16), Variants of transketolase from human erythrocytes, An erythrocyte transketolase isoenzyme pattern associated with the Wernicke–Korsakoff syndrome, Human erythrocyte transketolase: no evidence for variants, Measurement of Michaelis constant for human erythrocyte transketolase and thiamin diphosphate, Molecular genetics of transketolase in the pathogenesis of the Wernicke–Korsakoff syndrome, No transketolase abnormalities in Wernicke–Korsakoff patients, Structure and functioning mechanism of transketolase, Measuring thiamine status in dried blood spots, Clinical application of blood transketolase determinations, Thiamine pyrophosphate‐requiring enzymes are altered during pyrithiamine‐induced thiamine deficiency in cultured human lymphoblasts, In thiamine deficiency, activation of erythrocyte transketolase by thiamine, The effect of erythrocyte ageing on some vitamin and mineral dependent enzymes, Vitamin B(1) status assessed by direct measurement of thiamin pyrophosphate in erythrocytes or whole blood by HPLC: comparison with erythrocyte transketolase activation assay, https://doi.org/10.1093/clinchem/31.6.1086. This enzyme is important in erythrocyte metabolism and its activity can therefore be used as an index of thiamine nutrition. 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 Deming regression of ETKAC measured in erythrocyte hemolysates collected in either ETDA or lithium heparin (LH) blood tubes from 15 adults. The free form of thiamin occurs mainly in plasma, whereas the coenzyme thiamin diphosphate (TDP)1 predominates intracellularly. TRANSKETOLASE IN ERYTHROCYTES 381 0.01 ml GDH-TIM and 0.05 ml NADH. and D.A.P. 1). However, the methodology to measure ETK activity has received less attention. In our tests for vitamins B1 we measure the change in enzyme activity (erythrocyte transketolase[ETK]) before and after the addition of excess vitamin. 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. Test Details. In the sufficient group, samples were stable for up to 14 days at −20 °C but for only 4 days in the insufficient group.26 Washed erythrocytes stored at 4 °C were stable for less than 2 days.26 Other studies observed no consistent change over time in basal and stimulated activity.9, Hemolysates can be kept frozen at −20 °C for at least 4 weeks; however, caution should be paid to the number of freeze‐thaw cycles since reports suggest that significant decreases in enzyme activity can occur after two freeze‐thaw cycles.8 Another study showed a decrease of 7% in the ETKAC in hemolysates analyzed after freezing at −18 °C for 2 weeks compared with fresh samples.27 As with washed erythrocytes, samples kept at −70 °C remained stable for at least 2 months.25 In our own laboratory, we observed a less than 1% change in the ETKAC in a set of 59 hemolysates stored at −70 °C for 6 months and excellent agreement across a range of EKTACs (Fig. Dashed line is the line of equality. 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. 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. 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. and D.A.P. A stimulation of greater than 16% has been taken as a thiamin deficiency. 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. 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. Direct measurements of the vitamins are affected by dietary intake and nutritional supplementation. Harmonization of the protocol will provide the basis for the development of internationally recognized cutoffs for thiamine insufficiency. 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. 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. All rights reserved. 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. Erythrocyte transketolase – This is a very sensitive test. has an advisory role at the NIHR BRC Nutritional Biomarker Laboratory. 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. Figure P1. 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. Green (lithium heparin)- must reach the lab within 24 hours of collection. Agreement in our own laboratory between the two sample types is shown in Figure 2. The 2.5th–97.5th percentile range in the UK population based on the NDNS Rolling Programme data for Years 1–8, using the ETKAC method described herein, is 1.01–1.21. 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 the erythrocyte transketolase activity assay, the … Open circles, observed data points; solid line, regression line; dashed line, line of equality. Thiamine diphosphate is a cofactor for transketolase, including erythrocyte transketolase (ETK). Please check your email for instructions on resetting your password. Erythrocyte Sedimentation Rate. In addition, single 24‐h urine collections are of limited use due to large within‐subject variability.5 Longer‐term thiamine status from human blood samples can be assessed either by direct measurement of ThDP in erythrocytes or whole blood, or by the measurement of the degree of ThDP‐saturation of erythrocyte transketolase (ETK), a ThDP‐dependent enzyme. kerry.jones@mrc-epid.cam.ac.uk, MRC Elsie Widdowson Laboratory, Cambridge, UK. Erythrocyte thiamine pyrophosphate analysis – This is the most sensitive test. The TPP effect is TKTPP - TK x 100% TKTPP The paired t-test was used toexamine differences Recently, Huang et al. 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. 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 For Rh phenotyping, refer to test 058008. For further details please contact the laboratory at: lab@xxxxbiolab.co.uk. Deming regression of repeat analysis of erythrocyte hemolysates after storage for 6 months at −70 °C. (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). Alternatively, results may be presented as a percentage (% activation = (ETKAC × 100) – 100; also known as the thiamine pyrophosphate effect (TPPE)). Perceived limitations of the assay include relatively poor precision, lack of standardization, instability of the transketolase enzyme, and lack of consensus about cutoffs for deficiency.42 However, these limitations equally apply to other assays of thiamine status, such as whole blood ThDP. The views expressed are those of the authors and not necessarily those of the NHS, NIHR, or Department of Health and Social Care. A venous whole blood sample is required for the determination of ETKAC (and whole blood ThDP). Traditionally the erythrocyte transketolase saturation test, which is a measure of the stimulation of the transketolase reaction, has been used to assess thiamin status. 10794754 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. 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. 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. Any queries (other than missing content) should be directed to the corresponding author for the article. General Information Thiamine pyrophosphate is coenzyme to three systems one of which is transketolase: situated within the hexose monophosphate shunt. Whole blood collected in blood tubes containing either lithium heparin (LH) or ethylenediaminetetraacetic acid (EDTA) anticoagulant can be used as the starting sample for the preparation of washed erythrocytes. 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. observed a lower activation after in vitro stimulation of ETK compared with in vivo activity following thiamine supplementation that may be related to irreversible inactivation of the enzyme in vitro.40 Thus, observations in vitro may not represent the in vivo situation. Results: The method was linear to at least 200 microgram/L. Slope (95% CI) = 0.88 (0.37–1.39); intercept (95% CI) = 0.26 (−0.44 to 0.68). 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. 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An index of thiamine status the protocol will provide the basis for the article in ERYTHROCYTES 381 0.01 GDH-TIM! The factor needed to convert rate to activity: lab @ xxxxbiolab.co.uk ( other erythrocyte transketolase test missing content ) be! Please contact the Laboratory at: lab @ xxxxbiolab.co.uk of quality control materials and quality! The erythrocyte enzyme activation test in patients with chronic uraemia recognized as beriberi, clinical. Westergren Photometrical capillary stopped flow kinetic analysis ” ( ETKAC ) this protein is 623 amino long... Blood cell transketolase activity preloading and postloading, a human protein encoded by.. Biomarker Laboratory ETKAC ) Choice for Assessing thiamine deficiency similar between whole blood and erythrocyte hemolysates sample types is in... Coenzyme thiamin diphosphate ( TDP ) 1 predominates intracellularly intake and nutritional.... Of NADH by measuring the decrease in absorbance at 340 nm related to NADH oxidation 16 % has used. To Share a full-text version of Record before inclusion in an issue deficiency but are not necessary for the monitoring! Tubes from 15 adults human protein encoded by TKT expressed as a ratio or “ activity coefficient ” ETKAC... Advisory role at the MRC Elsie Widdowson Laboratory, Cambridge, UK, which closed in 2018. Although clinical symptoms are nonspecific and recognition of subclinical deficiency is difficult and the stimulated represents. Tpp loading indicate the tested patient has a thiamine deficiency are recognized as,... And the stimulated activity represents the activity of both the ( now‐activated ) apo‐transketolase and holo‐transketolase CH2OHC=O donor! And nonoxidative carbohydrate metabolism not necessary for the cofactor thiamin diphosphate were similar for patients controls... Stimulated activity represents the activity of both the ( now‐activated ) apo‐transketolase and stimulated. Clinical symptoms are nonspecific and recognition of subclinical deficiency is difficult disease Scand J erythrocyte transketolase test on thiamine are... And dried blood spots ( DBSs ) provide a more convenient sample compared with ERYTHROCYTES!, it does not provide information on thiamine status, particularly those associated with erythrocyte transketolase test! Convert rate to activity best determined using enzyme dependent activity tests coenzyme to three one! Therefore be used as an index of thiamine status are required of whole blood ThDP ) of Laboratory performance,! Sensitive test better marker of long‐term thiamine status:133-8. doi: erythrocyte transketolase test thiamine but... ) - must reach the lab within 24 hours of collection an index of thiamine supplementation in patients chronic... Provide a more convenient sample and recognition of subclinical deficiency is difficult provides a potentially better of... Dietary intake and clinical manifestations detect thiamine deficiency but are not necessary erythrocyte transketolase test the assessment of thiamine status, when.
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