Introducing a New and Improved Thiamine Testing Platform

Introduction

It is my great to pleasure to introduce a new thiamine testing platform, the Erythrocyte Transketolase Activity Coefficient (ETKAC), offered by the Clinical Immunology Laboratory, in North Chicago, IL. ETKAC is a functional enzymatic test designed to evaluate the activity of the thiamine-dependent enzyme, erythrocyte transketolase, both in its resting and activated state. Compared to other available thiamine testing assays, the ETKAC provides functional assessment of in-cell activated thiamine and evaluates potential abnormalities in thiamine-dependent enzymes. The test is currently available via physician request, but we hope to provide more direct access in the future.

What Is Thiamine Deficiency and Why We Need Better Testing

Thiamine (vitamin B1) is the precursor to the coenzyme thiamine pyrophosphate (TPP), also termed thiamine diphosphate (TDP/ThDP). It is an essential B vitamin in humans that is required for carbohydrate, fatty acid, and even protein metabolism. As such, it is critical for health and plays an important role in nerve function.

TPP, the bioactive component of thiamine, drives enzymes found in multiple metabolic pathways that include the pentose phosphate pathway, citric acid cycle, and glycolysis. In this way, thiamine contributes to the structure of the nervous system by inducing energy production (ATP), and synthesis of vital compounds such as lipids and acetylcholine. Without thiamine, mitochondrial respiration is suppressed and ATP capacity wanes.

Frank or severe deficiencies in thiamine result in Beriberi and Wernicke-Korsakoff Syndrome. These syndromes are well understood and, in some cases, can be attributed to alcoholism and malnutrition. Despite being well understood, frank deficiency may be missed clinically, especially in non-alcoholic and presumably well-nourished populations, but also in the early stages of the deficiency when symptoms may be attributed to other common disease processes. Some of the patient populations with underlying thiamine deficiency who may benefit from thiamine testing include those diagnosed or suspected of chronic fatigue syndrome, fibromyalgia, individuals who are diabetic, or who are experiencing neurocognitive or neuromuscular symptoms. Pregnant women should also be tested for deficiency, especially when hyperemesis gravidarum or pre-eclampsia are present. More information on patient populations affected by insufficient thiamine can be found here.

Unfortunately, traditional testing may miss subclinical, functional, and genetically-induced thiamine deficiencies. Plasma testing, for example, is highly susceptible to dietary fluctuations in thiamine. If an individual has recently consumed thiamine-rich foods, the test may indicate thiamine sufficiency, when in reality, the individual is truly deficient. Likewise, if the individual is deficient in magnesium, which is required to activate thiamine (phosphorylate free thiamine into the bioactive thiamine pyrophosphate or TPP) and/or the individual has a genetic defect that diminishes the activity of any portion of the thiamine metabolic pathway, plasma thiamine tests will appear normal even though the individual is deficient. With magnesium deficiency, free thiamine may be sufficient, but bioactive thiamine will be deficient. Similarly, with thiamine-related genetic variants, free thiamine and perhaps even TPP will be within range, but the ability to use thiamine effectively will be impaired.

Whole blood TPP testing, although more accurate than plasma-based assessment and not susceptible to fluctuations in dietary intake, is still problematic in some cases. It is susceptible to supplemental intake [Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, pg 661], which means that if the individual is supplementing with thiamine either alone or in a daily multi-vitamin, and/or is on a thiamine-repletion protocol due to a recognized deficiency, the testing may falsely indicate that he/she is no longer deficient, despite symptoms to the contrary. Since whole blood TPP assess the bioactive form of thiamine, TPP, it is more sensitive to some functional deficiencies. Unfortunately, it is not sensitive to whether or not the individual has the capacity to use that TPP because of genetic abnormalities or chronic health issues.

Measuring Enzyme Activity to Assess Nutrient Status

Rather than measure the nutrient in circulation, a more accurate form of testing involves the measurement of enzyme activity in erythrocytes or red blood cells. Since enzymes depend upon specific vitamins for functionality, when we measure the enzyme activity both in its basal state (resting/uninfluenced) and in response to the nutrient, we can reliably assess nutrient status. This will more accurately portray tissue concentrations than circulating concentrations. In that regard, enzyme activity tests are considered functional assessments of nutrient status.

Enzyme activity tests have been used for decades to assess a number vitamins and minerals. For example, glutathione reductase activity is used to measure riboflavin (vitamin B2) status. Similarly, the enzyme transaminase is used to measure vitamin B6 activity. Minerals like zinc and magnesium are best evaluated intracellularly instead of just measuring circulating levels.

To measure thiamine, the erythrocyte transketolase test is used. It was initially developed around 1962 and further improved and utilized through the 1970s. However, as time passed, the assay fell out of use in favor of the quicker and more cost effective, though less clinically sensitive, plasma and whole blood measures.

Recognizing the increased incidence of modern thiamine deficiency and a need for more sensitive testing, the scientists at the Nutritional Biomarker Laboratory (NBL), in the University of Cambridge, have developed an improved erythrocyte transketolase activity coefficient, or EKTAC, test. A slightly modified form of the NBL assay has been validated and applied at the lab in which I co-direct,  Clinical Immunology Laboratory (CIL), in North Chicago, IL.

Evaluation of Thiamine via Erythrocyte Transketolase Activity

The transketolase enzyme is a TPP-dependent enzyme that can be found in the cytoplasm of a variety of tissues including blood cells and the liver. The Erythrocyte Transketolase Activity Coefficient (ETKAC) measures tissue level TPP as a function of transketolase ratio with and without the presence of exogenous TPP. That is, enzyme activity is assessed in its basal state and after TPP is added. If enzyme activity increases in the presence of TPP, it indicates deficiency. How much enzyme activity increases tells us how bad the deficiency is. Table 1. below shows the cutoff values between sufficiency and deficiency. The ETKAC range is well established in the literature and by the consensus of the clinical chemistry associations (AACC/ADLM).

Table 1. ETKAC reference ranges.

When a Normal Transketolase Test May Not Be Normal

While the measurement of the transketolase activity in response to thiamine is among the most sensitive and specific tests of thiamine deficiency at the tissue level and its results tell us whether the individual is able to use circulating thiamine effectively, there are caveats. There are instances where transketolase activity in response to thiamine will appear normal or near normal (ETKAC values close to 1.0), but clinical symptoms and basal activity of the enzyme suggest problems with thiamine. This is because enzyme kinetics have been altered either genetically or environmentally. Some examples of conditions that alter enzyme kinetics include:

1. Genetic mutations: Some mutations in the transketolase enzyme cause a lower affinity of the enzyme to TPP. Here, the affected individual will show falsely normal ETKAC but low basal activity (i.e. without addition of exogenous TPP). In this case, the clinician can use this information to manage these individuals and potentially embark on genetic testing, when indicated by paying closer attention to enzyme basal activity.
2. Reduced transketolase levels. Individuals with chronic low levels of thiamine can undergo reduction of transketolase levels. This may show up as normal ETKAC but low basal activity. This effect seems to correct upon repletion of thiamine. In this case, an individual will have a normal ETKAC (close to 1.0) that eventually increases as transketolase enzyme levels increase; thus the deficiency is unmasked. This happens because of the increased expression of apoenzyme (non-thiamine bound enzyme) without concomitant increases of sufficient thiamine concentrations. After this, as the patient is more replete with thiamine, ETKAC corrects back to true normal levels near 1.0.
3. Increased ETKAC. Some patients with bronchial and breast carcinomas may have falsely elevated ETKAC. It is thought that this is likely due to conversion issues from thiamine to its TPP forms (active co-enzyme). Use of basal activity parameter in this case will assist in excluding the possibility of aberrant enzyme expression.
4. Additional nutrient deficiencies. Other nutrient deficiencies may affect total enzyme levels. For example, Vitamin D has been shown to increase the expression of transketolase by close to 4 fold (400%) in some in vitro studies. Similarly, if zinc or other gene expression factors are needed for transketolase transcription, then the same effect from the above point would occur until these factors are first addressed.
5. Clinical conditions and medications. There are known clinical conditions (e.g. liver disease, uremic neuropath, gastrointestinal dysfunction, polyneuritis, diabetes), and drugs that can reduce the levels of the apoenzyme.

It is for these conditions and potentially others, that assessing and reporting the basal activity of the enzyme, along with its activation quotient is useful.

How to Interpret Basal Activity Results

Although the cut-offs for basal activity are not as clearly understood as those for the ETKAC test, a value of 0.59 U/gHb or less has been shown to indicate thiamine deficiency across different patient populations. The Clinical Immunology Laboratory group has recently confirmed the lower limit of 0.59 U/gHb, and established an upper normal limit of 1.00 U/gHb.

Table 2. Proposed ranges for ETK basal activity tests.

Conclusion

Thiamine, in its active form TPP, functions as a rate-limiting co-enzyme in multiple pathways related to carbohydrate and energy metabolism. Deficiency of thiamine results in a host of seemingly unconnected symptoms that range in severity depending on the degree and duration of deficiency. It is not uncommon for frank and subclinical thiamine deficiency to be missed due to the cluster of seemingly unconnected symptoms, but also because of unavailability of sensitive and accurate testing. The ETKAC assay has an advantage to other thiamine assays due to it being a functional enzymatic test that can detect transketolase abnormalities, as well as thiamine status, as discussed above. In contrast, directly measuring plasma thiamine or whole blood TPP can lead to false normal results or miss a functional deficiency, respectively.

The ETKAC and Basal Activity assays have not been readily available in the United States for clinical testing until now. The Clinical Immunology group in North Chicago, a high-complexity CLIA-approved laboratory, has recently validated an affordable, and easy to order ETKAC assay. The test currently requires ordering through a physician; however the laboratory is planning on providing a direct-to-consumer service in the near future. CIL’s ETKAC assay provides both quotient and Basal Activity parameters in order to give patients and doctors the results they need for appropriate treatment. More information about the test, the requisition and sample shipping instructions can be accessed through CIL’s webpage. CIL can be contacted for questions regarding ETKAC ordering and/or interpretation. More information about ETKAC can be found on the laboratory’s website.

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Photo by Louis Reed on Unsplash.