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Title: A biochemical genetic investigation of transketolase in the Wernicke-Korsakoff syndrome
Author: Al-Bustan, Suzanne Ahmad Abdulbaqi
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1992
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Abstract:
The Wernicke-Korsakoff syndrome (WKS) is a severe neuropsychiatric disorder most commonly seen as a result of malnutrition associated with chronic alcohol abuse. In its acute form (Wemicke's encephalopathy, WE) it is characterised by ophthalmoparesis, nystagmus, ataxia and an apathetic confusional state. In those that survive this initial phase, over 70% will be left with a specific form of memory impairment with a varying degree of severity. In those most severely affected (~50% Korsakoff s psychosis, KP), the impairment will be such that they are incapable of independent living and many will require long-term institutional care. In Scotland there is good evidence of an increase, over the past two decades, in the number of people suffering from alcohol-related brain damage, of which KP is the major form (Smith & McColl, 1992). As a result the absolute number of patients in long term psychiatric care and suffering from alcoholic KP has grown. They form a small but significant proportion of beds in the refractory wards of mental hospitals. One general argument that has fuelled interest in the possibility of genetic factors in alcoholic KP is the fact that only a small percentage of those who chronically abuse alcohol and become malnourished ever develop the characteristic neuropathology. In 1930 Wagner and Weir reported on the association of the WKS with thiamine depletion. Thiamine exists in three forms, thiamine monophosphate, triphosphate and pyrophosphate (TPP): the last making up 80-90% of total thiamine in cells. TPP is a cofactor for many enzymes of carbohydrate metabolism including pyruvate dehydrogenases, alpha-ketoglutarate dehydrogenase and transketolase (TK). Several studies have implicated TK in the pathogenesis of the WKS. The aim of this study was to investigate the role of TK in the WKS using quantitative and qualitative methods of analysis in patient and control groups. Blood from 30 hospitalized patients with the WKS and 25 normal controls was analysed for erythrocyte transketolase (ETK) activity and stimulated erythrocyte transketolase (ETKs) activity by the addition of TPP using an end point assay. By employing a modified TK assay (Smeets et al., 1971; Bayoumi & Rosalki, 1976), mean ETK activity in 10 normal controls was found to be 0.418 +/- 0.193 lU/mg protein which was not significantly different (p > 0.1) from the 20 patients (mean ETK activity 0.406 +/- 0.155 lU/mg protein). The same was true for the ETKs activity in which normal controls had a mean of 0.738 +/- 0.208 IU/mg protein and patients 0.680 +/- 0.190 IU/mg protein. This difference was not statistically significant (p > 0.1). A report (Kaczmarek & Nixon, 1983) suggesting the existence of several species of TK was investigated by polyacrylamide gel electrophoresis (PAGE). TK activity was detected on PAG by specific TK enzyme stain. All normal controls and patient samples were run on PAG and then stained both specifically and generally (Coomassie-Brilliant Blue (CBB) R-250 and silver stain). They yielded two ETK bands which varied in intensity and were cathodal to haemoglobin. Although qualitative analysis strongly suggested ETK to be heterogeneous, it did not provide means of investigating WKS by biochemical analysis. Because the analysis of ETK (quantitatively and qualitatively) showed no apparent differences between the two groups, it was further investigated following the purification of the enzyme. PAGE crosslinked with DHEBA, a form of crosslinker which allows gels to be solubilized under alkaline conditions, provided means of isolating pure fractions of TK from red blood cells of normal controls and patients. These extracted fractions were subjected to analysis using high performance liquid chromatography (HPLC), isoelectric focusing (lEF) and endpoint assay. Despite confirmation of TK heterogeneity (with respect to ionic strength, isoelectric point (pi) and ETK activity), no differences in electrophoretic profile between patients and controls were revealed. In all samples two distinct bands of varying intensity appeared at pi 7.35 and 6.55 on BEF gels. Further studies on native TK were carried out after purification from white blood cells which had been collected as buffy coats from normal individuals (Mocali & Paoletti, 1989). Enzyme recovery declined as the steps of purification progressed, therefore another purification method was developed from the method of Takeuchi et. al.(1986) (where enzyme recovery was not sufficient for amino acid analysis). The fractions isolated by this method were subjected to fast protein liquid chromatography (FPLC) on Mono Q column which yielded two peaks containing TK activity. The fractions were also loaded on an SDS-PAGE where the protein band had a molecular weight (MW) of 68kD (kiloDaltons). These results suggested that TK exists in at least two forms varying in ionic strength as revealed by the chromatographic profile from FPLC.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796709  DOI: Not available
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