Thiamine (vitamin B1) is important for the metabolism of carbohydrates and maintenance of the nervous system. Thiamine is required by the body in the form of thiamine diphosphate (TDP). TDP is a co-factor for transketolase, an enzyme in the pentose phosphate shunt which generates NADH necessary for anabolic biosynthetic reactions. Thiamine stores are relatively small therefore deficiencies can develop quickly particularly in chronic alcoholics, the elderly and patients requiring parenteral nutrition. As a thiamine deficiency develops the levels of TDP in erythrocytes and brain tissue remain stable until stores become significantly deplete. Most circulating TDP (up to 90%) is found in erythrocytes. A decrease in thiamine levels in erythrocytes correlates with a decrease in the TDP in other tissues. A clinical consequence of long term alcohol abuse is low thiamine levels which is implicated in the aetiology of Wernicke’s encephalopathy (WE) with cases confirmed in 2% of autopsies in western society.
Currently, nutritional status of thiamine may be assessed in the laboratory by direct measurement of TDP in whole blood using HPLC, or less commonly by measurement of the functional marker erythrocyte transketolase activity (ETKA) in the absence or presence of TDP in vitro. While direct measurement of thiamine in whole blood has been shown to be helpful in the initial assessment of thiamine status, there has been some questions over its utility in monitoring the efficacy of treatment. Historically, ETKA has been measured using a centrifugal analyser with spectrophotometric measurement of NADH consumption. Here we describe the adaptation and optimisation of a semi-automated method for the measurement of ETKA for use with a BioTek Synergy H1 plate reader. ETKA will be used to assess thiamine status and efficacy of treatment in chronic alcoholic patients and compared to direct measurement of TDP.
We hypothesise that ETKA may be more useful monitoring patients who undergoing treatment. ETKA requires TDP and a divalent cation for its activity. Magnesium status in chronic alcoholics is assessed by measuring serum magnesium, however this only reflects around 1% of total body magnesium. A robust method for measuring erythrocyte magnesium by ICP-MS will be validated and verified. This will be used to establish if whole body magnesium stores are deplete in chronic alcoholic patients so we hypothesise that erythrocyte magnesium may be a better marker of total body status than serum magnesium.
A prospective randomised controlled trial will assess the effect of magnesium sulphate or Pabrinex® administration on ETKA and erythrocyte magnesium in alcohol dependent patients at risk of Wernicke’s Korsakoff Syndrome. This trial will aim to answer whether magnesium administration enhances the biochemical response to thiamine as measured by the activity of the enzyme transketolase in erythrocytes.