Impairment of acetylcholine synthesis in thiamine deficient rats developed by prolonged tea consumption

The synthesis of whole brain acetylcholine is reduced in thiamine deficient rats produced by prolonged administration of tea. In those rats fed a normal diet and given tea (1:50, w/v) instead of drinking water for 20 weeks, the conversion of [14C] pyruvate to [14C]acetylcholine decreased by 35%. However, no neurological symptoms were observed. Administration of tea to rats fed a thiamine half-deficient diet for 7-8 weeks caused not only 60% decrease in acetylcholine synthesis but also neurological symptoms. This decreased synthesis of acetylcholine is related to a decline in pyruvate dehydrogenase activity. The results suggest that prolonged administration of tea to rats cause an impairment of acetyl CoA production resulting in a deficit in acetylcholine synthesizing capacity.     

Effect of thiamine on the activity of the pyruvate dehydrogenase complex in rat liver following stimulation of lipogenesis

It is shown that thiamine administration to rats (250 micrograms per 100 g of mass) who were given high-carbohydrate diet (lipogenesis intensification) after fasting inhibits an increase in the pyruvate dehydrogenase activity in the liver homogenate and mitochondria usual under these conditions. This is observed when determining total activity of the pyruvate dehydrogenase complex and activity of its first component--pyruvate dehydrogenase estimated from the ferricyanide reduction and [1-14C] CO2 formation from [1-14C] pyruvate. Fasting animals and animals whom thiamine was administered against a background of lipogenesis intensification revealed a higher ability of the liver tissue to synthesize acetoin as compared with the control group and animals with the intensified lipogenesis without thiamine administration.     

Paraquat and menadione exposure of rainbow trout (Oncorhynchus mykiss)--studies of effects on the pentose-phosphate shunt and thiamine levels in liver and kidney

Possible xenobiotic interactions with thiamine were studied in salmonid fish, by repeatedly injecting two model substances, paraquat and menadione, into juvenile rainbow trout (Oncorhynchus mykiss). These two substances were chosen because of their well-known ability to redox-cycle and cause depletion of NADPH in several biological systems. Depletion of NADPH increases metabolism through the pentose-phosphate shunt and may thereby increase the need for thiamine diphosphate by heightened transketolase activity. A special food was produced with lower thiamine content than commercial food, usually enriched with thiamine, which could mask an effect on the thiamine level. After 9 weeks of exposure, glucose-6-phosphate dehydrogenase, transketolase, glutathione reductase and ethoxyresorufin O-deethylase were analysed in liver and kidney cellular sub-fractions as well as analysis of total thiamine concentrations in liver, kidney and muscle. The results showed that paraquat caused a large increase in hepatic glutathione reductase activity and induced hepatic glucose-6-phosphate dehydrogenase activity, i.e., the rate-limiting enzyme in the oxidative part of the pentose-phosphate shunt. Despite this paraquat exposure did not affect transketolase activity and total thiamine concentration.     

Enzyme activity of thiamine pyrophosphate in the rat after oxythiamine administration

Intraperitoneal injection of hydroxythiamine to rats (1 mmol per kg bw) resulted after 2-4 h in a more than 4-fold decrease in the activity of the oxoglutarate dehydrogenase complex, pyruvate dehydrogenase complex and NADP-dependent isocitrate dehydrogenase in adrenal mitochondria. Inhibition of hyaloplasmic transketolase, 6-phosphogluconate dehydrogenase and NADP-dependent malate dehydrogenase occurred later. Based on the correlation of the time course of enzymatic activity in the adrenals and the decreased concentration of 11-hydroxycorticosteroids in the blood the paramount role in the maintenance of the steroidogenesis among thiamine pyrophosphate-containing enzymes is assigned to the oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes.     

Effect of thiamine phosphates on the activity of regulatory enzymes of the pyruvate dehydrogenase complex

The effect of thiamine triphosphate (ThTP) and thiamine diphosphate (ThDP) on the activity of rat liver pyruvate dehydrogenase complex regulatory enzymes (kinase and phosphatase) was studied in experiments with isolated enzyme preparations. It is shown that ThDP caused a pronounced activation of pyruvate dehydrogenase phosphatase (Ka is equal to 65.0 nM). ThTP inhibits phosphatase competitively against the substrate--the phosphorylated pyruvate dehydrogenase complex. The both thiamine phosphates inhibit the pyruvate dehydrogenase kinase activity almost similarly in concentrations exceeding 10 microM. The physiological significance of the antagonistic action of ThDP and ThTP on the pyruvate dehydrogenase phosphatase activity is discussed.     

Regional alterations of thiamine phosphate esters and of thiamine diphosphate-dependent enzymes in relation to function in experimental Wernicke's encephalopathy

Thiamine phosphate esters (thiamine monophosphate-TMP; thiamine diphosphate-TDP and thiamine triphosphate-TTP) were measured as their thiochrome derivatives by High Performance Liquid Chromatography in the brains of pyrithiamine-treated rats at various stages during the development of thiamine deficiency encephalopathy. Severe encephalopathy was accompanied by significant reductions of all three thiamine phosphate esters in brain. Neurological symptoms of thiamine deficiency appeared when brain levels of TMP and TDP fell below 15% of normal values. Activities of the TDP-dependent enzyme -ketoglutarate dehydrogenase were more severely reduced in thalamus compared to cerebral cortex, a less vulnerable brain structure. On the other hand, reductions of TTP, the non-cofactor form of thiamine, occurred to a greater extent in cerebral cortex than thalamus. Early reductions of TDP-dependent enzymes and the ensuing metabolic pertubations such as lactic acidosis impaired brain energy metabolism, and NMDA-receptor mediated excitotoxicity offer rational explanations for the selective vulnerability of brain structures such as thalamus to the deleterious effects of thiamine deficiency.     

Effects of thiamine deprivation on the growth and metabolism of the phytoflagellatePolytomella agilis

The effects of thiamine deprivation on the growth, respiration, and activity of several enzymes of the phytoflagellate protozoanPolytomella agilis were studied. Vitamin deprivation had no effect on the exponential growth rate; the peak population of cultures grown without thiamine was 50% of the control level. The rates of oxygen consumption in control and thiamine-deprived cultures were not significantly different from each other. The activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase in vitamin-deprived cells were 14% and 30%, respectively, of the control values. In these cells, the succinic dehydrogenase activity was 10% and mitochondrial ATPase activity was twice that of control cells. Vitamin deprivation had no effect on the activities of malate dehydrogenase and isocitrate lyase, but pyruvic carboxylase activity increased fourfold. These results indicate a complex role for thiamine in the regulation of growth, respiration, and metabolism in this organism.     

Effects of maternal thiamine deficiency on the development of thiamine-dependent enzymes in regions of the rat brain

Previous studies suggest that developing rat brain is susceptible to reduced thiamine intake. In order to assess the metabolic basis for this susceptibility, activities of three thiamine-dependent enzymes (pyruvate dehydrogenase complex, -ketoglutarate dehydrogenase and transketolase) were measured in homogenates of brain tissue from the offspring of thiamine-deficient mothers. Control groups of animals were pair-fed to equal food consumption with the thiamine-deficient animals. The study revealed region-selective delays in the establishment of adult activities of thiamine-dependent enzymes as a result of maternal thiamine deficiency. Pyruvate dehydrogenase complex activities in cerebral cortex were significantly reduced (by 20% P < 0.05); -ketoglutarate dehydrogenase activities were also reduced in cerebral cortex (by 30% P < 0.05). In the case of transketolase, enzyme activities were significantly reduced in cerebral cortex, cerebellum and brainstem. Following thiamine replenishment, defective enzyme activities were restored to normal in all cases. However, since thiamine-dependent enzymes are important for the establishment of adult patterns of cerebral energy metabolism and also in myelin synthesis, maternal thiamine deficiency resulting in reductions of thiamine-dependent enzymes at a vulnerable period in brain development could have serious metabolic consequences leading to permanent neurological sequellae in the offspring.     

Postnatal development of thiamine metabolism in rat skeletal muscle.

1. The activities of 2-oxoglutarate dehydrogenase, transketolase, thiamine pyrophosphokinase and thiamine triphosphatase and the concentrations of thiamine phosphates were almost the same between rat extensor digitorum longus and soleus muscles at 2 weeks of age. 2. These enzyme activities changed after 3 weeks of age in a different way depending on the muscle phenotype. 3. Thiamine diphosphate level and the activity of 2-oxoglutarate dehydrogenase increased only in soleus muscle and thiamine triphosphate level increased only in extensor digitorum longus during development.     

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.     

Role of thiamine thiol form in nitric oxide metabolism

In alkaline media the thiamine cyclic form is converted into a thiol form (pK(a) 9.2) with an opened thiazole ring. The thiamine thiol form releases nitric oxide from S-nitrosoglutathione (GSNO). Thiamine disulfide, mixed thiamine disulfide with glutathione, and nitric oxide are produced in the reaction. Free glutathione was recorded in small amounts. The concentration of formed nitric oxide agreed well with the concentration of degraded GSNO. The concentration of released nitric oxide was determined under anaerobic conditions spectrophotometrically by production of nitrosohemoglobin. In air, the release of nitric oxide was recorded by the production of nitrite or the oxidation of oxyhemoglobin to methemoglobin. The concentration of the thiol form in the body under physiological pH values (7.2-7.4) did not exceed 1.5-2.0%. We believe that due to the exchange reactions between the thiamine thiol form and S-nitrosocysteine protein residues, nitric oxide can be released and mixed thiamine-protein disulfides are formed. The mixed thiamine disulfides (including thiamine ester disulfides) as well as the thiamine disulfide form are quite easily reduced by low molecular weight thiols to form the thiamine cyclic form with a closed thiazole ring. A possible role of the thiamine thiol form in releasing deposited nitric oxide from low-molecular-weight S-nitrosothiols and protein S-nitrosothiols and in regulation of blood flow in the vascular bed is discussed.     

Characteristics of carbohydrate metabolism in the rat liver in thiamine deficiency

Thiamine deficiency in rats induced by oxythiamine is accompanied by an increase in the free NADP+/NADPH ratio in liver tissue, which results in multifold stimulation of the metabolite flux in the oxidation branch of the pentose cycle. The increase in the intracellular concentrations of isocitrate and alpha-ketoglutarate with a simultaneous decrease of malate in the liver of vitamin-deficient rats points to the inhibition of alpha-ketoglutarate dehydrogenase responsible for the anomalous metabolism under conditions of thiamine deficiency. The decrease of the functional activity of the tricarboxylic acid cycle is concomitant with the activation of conversions in the oxidation branch of the pentose cycle, glucuronate and glycolytic pathways of carbohydrate metabolism, which is directed at eliminating the energy deficiency in rats with B1-hypovitaminosis.     

The effect of thiamine and its metabolites on the activity of tissue and purified lactate dehydrogenase

It is shown that thiamine and its metabolites effect lactate dehydrogenase activity and lactate content in the tissues. Thiochrome and thiamine phosphate increase the lactate level in the liver and small intestine. The given effect correlates with the inhibition of the tissue and purified lactate dehydrogenase by thiochrome.     

Energy and glucose pathways in thiamine deficient primary rat brain microvascular endothelial cells

Thiamine deficiency (TD) results in lactate acidosis, which is associated with neurodegeneration. The aim of this study was to investigate this alteration in primary rat brain endothelia. Spectrophotometric analysis of culture media revealed that only a higher concentration of pyrithiamine, which accelerates the intracellular blocking of thiamine, significantly elevated the lactate level and lactate dehydrogenase activity within 7 days. The medium without pyrithiamine and with a thiamine concentration comparable to pathophysiological plasma levels mildly reduced only the activity of transketolase. This suggests that significant metabolic changes may not occur at the early phase of TD in cerebral capillary cells, while anaerobic glycolysis in capillaries may be mediated during late stage/chronic TD.     

A constitutive thiamine metabolism mutation, thi80, causing reduced thiamine pyrophosphokinase activity in Saccharomyces cerevisiae.

We identified a strain carrying a recessive constitutive mutation (thi80-1) with an altered thiamine transport system, thiamine-repressible acid phosphatase, and several enzymes of thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole. The mutant shows markedly reduced activity of thiamine pyrophosphokinase (EC 2.7.6.2) and high resistance to oxythiamine, a thiamine antagonist whose potency depends on thiamine pyrophosphokinase activity. The intracellular thiamine pyrophosphate content of the mutant cells grown with exogenous thiamine (2 x 10(-7) M) was found to be about half that of the wild-type strain under the same conditions. These results suggest that the utilization and synthesis of thiamine in Saccharomyces cerevisiae is controlled negatively by the intracellular thiamine pyrophosphate level.     

Enhanced hepatotoxicity and inhibition of liver endoplasmic reticulum calcium pump by CCl4 in rats fed a thiamine deficient diet

Male Sprague-Dawley rats were fed a thiamine deficient diet for three weeks, then treated with a range of CCl4 doses (0.01-1-ml/kg). Rats fed the deficient diet grew more slowly (body weight 65 percent of control) and had elevated liver glutathione (GSH) (220 percent of control). CCl4 hepatotoxicity, assessed by serum glutamicpyruvic transaminase (SGPT) activity and histological examination 24 hours after the hepatotoxin, was augmented in the group fed the thiamine deficient diet. Likewise, CCl4 inhibition of liver endoplasmic reticulum (ER) function (glucose-6-phosphatase (G6Pase) and calcium pump activities one hour after CCl4) was enhanced in rats fed the deficient diet. These results suggest that thiamine deficiency enhances CCl4 damage to membranes of the ER and enhances CCl4 hepatotoxicity.     

Half-of-the-site reactivity of the decarboxylating component of the pyruvate dehydrogenase complex from pigeon breast muscle with respect to 2-hydroxyethyl thiamine pyrophosphate

The holopyruvate dehydrogenase is characterized by the charge transfer complex formation between tryptophan residue and thiamine pyrophosphate in each of two active centres. Interaction of apoenzyme with one mole of 2-hydroxyethyl thiamine pyrophosphate results in appearance of the same spectral band which does not change in intensity with further increase in ligand concentration. 2-hydroxyethyl thiamine pyrophosphate: acceptor oxidoreductase activity abolishes after oxidation of only one tryptophan residue per mole of the protein or blocking of one of the active centres with inactive analogue of the coenzyme. In the latter case the charge transfer complex band induced by interaction of apoenzyme with 2-hydroxyethyl thiamine pyrophosphate was not shown at all. These facts testify to half-of-the-site reactivity of pyruvate dehydrogenase with respect to 2-hydroxyethyl thiamine pyrophosphate.     

Reversibility of thiamine deficiency-induced partial necrosis and mitochondrial uncoupling by addition of thiamine to neuroblastoma cell suspensions

Culture of neuroblastoma cells in the presence of low thiamine concentration (6 nM) and of the transport inhibitor amprolium leads to the appearance of signs of necrosis: the chromatin condenses, the oxygen consumption decreases and is uncoupled, the mitochondrial cristae are disorganized, the thiamine diphosphate-dependent dehydrogenase activities are impaired. When 10 µM thiamine are added to these cells, the basal respiration increases, the coupled respiration is restored and mitochondrial morphology is recovered within 1 h. Addition of succinate, which is oxidized via a thiamine diphosphate-independent dehydrogenase, to digitonin-permeabilized cells immediately restores a coupled respiration. Our results suggest that the slowing of the citric acid cycle is the cause of the biochemical lesion induced by severe thiamine deficiency and that part of the mitochondria remain functional. (Mol Cell Biochem 174: 121–124, 1997)     

Tricarboxylic acid cycle enzymes following thiamine deficiency

Thiamine (Vitamin B1) deficiency (TD) leads to memory deficits and neurological disease in animals and humans. The thiamine-dependent enzymes of the tricarboxylic acid (TCA) cycle are reduced following TD and in the brains of patients that died from multiple neurodegenerative diseases. Whether reductions in thiamine or thiamine-dependent enzymes leads to changes in all TCA cycle enzymes has never been tested. In the current studies, the pyruvate dehydrogenase complex (PDHC) and all of enzymes of the TCA cycle were measured in the brains of TD mice. Non-thiamine-dependent enzymes such as succinate dehydrogenase (SDH), succinate thiokinase (STH) and malate dehydrogenase (MDH) were altered as much or more than thiamine-dependent enzymes such as the alpha-ketoglutarate dehydrogenase complex (KGDHC) (-21.5%) and PDHC (-10.5%). Succinate dehydrogenase (SDH) activity decreased by 27% and succinate thiokinase (STH) decreased by 24%. The reductions in these other enzymes may result from oxidative stress because of TD or because these other enzymes of the TCA cycle are part of a metabolon that respond as a group of enzymes. The results suggest that other TCA cycle enzymes should be measured in brains from patients that died from neurological disease in which thiamine-dependent enzymes are known to be reduced. The diminished activities of multiple TCA cycle enzymes may be important in our understanding of how metabolic lesions alter brain function in neurodegenerative disorders.     

Activities of thiamine-dependent enzymes in two experimental models of thiamine deficiency encephalopathy: 3. Transketolase

Chronic thiamine deprivation in the rat leads to ataxia, loss of righting reflex and neuropathological damage to lateral vestibular nucleus. Before onset of neurological symptoms, transketolase (TK) activities were found to be selectively reduced by 25% in lateral vestibular nucleus and surrounding pons. Further progression of thiamine deprivation resulted in a generalized reduction in TK activity. Measurement of enzyme activity in the presence of added TPP cofactor in vitro did not lead to normalisation of enzyme activities suggesting loss of apoenzyme. Administration of thiamine to symptomatic thiamine-deprived rats resulted in reversal of neurological symptoms and to normalisation of defective TK activities in less vulnerable structures such as cerebral cortex striatum and hippocampus; reduction of TK activity, however, persisted in brainstem and cerebellar regions. Pyrithiamine treatment results, within 3 weeks, in loss of righting reflex, convulsions and more widespread neuropathological damage compared to that observed following thiamine deprivation. TK activity was found to be significantly decreased before the onset of neurological symptoms in all brain regions and appearance of symptoms was accompanied by more severe reductions of TK. In contrast to chronic thiamine deprivation, TK activities following pyrithiamine treatment were: (i) equally reduced in magnitude in vulnerable and non-vulnerable brain structures, (ii) unchanged following reversal of neurological abnormalities by thiamine administration.