Tesi sul tema "Thiamine deficiency"

Pyrithiamine-induced thiamine deficiency (PTD) in the rat results in a metabolic disorder associated with selective cerebral vulnerability. The basis of the histological lesions that develop in areas that include the thalamus, medial geniculate, and inferior colliculus is undetermined but an underlying glutamate-mediated form of excitotoxicity may be responsible. This work examines the possibility that PTD leads to changes in extracellular glutamate concentration, membrane polarization status, and gene expression consistent with a decreased ability to maintain homeostatic integrity.
At the acute symptomatic stage, extracellular glutamate by concentration as determined microdialysis was found to be elevated in the thalamus but within normal limits in the non-vulnerable parietal cortex. This result suggests an involvement of glutamate in the pathogenesis of thiamine deficiency lesions. An in vivo examination of L-type voltage-sensitive calcium channel activity using quantitative autoradiography and ($ sp3$H) -nimodipine at the same stage of PTD revealed that increased activity was present in and localized to the thalamus, but was absent in areas resistant to histological damage. The finding indicates a likelihood of regional depolarization. Finally, induction of the proto-oncogene c-fos was detected in the thalamus and inferior colliculus with quantitative in situ hybridization analysis at the acute symptomatic stage, thereby confirming the association of PTD with depolarization-related events occurring in advance of the appearance of frank infarction.
Together, these results suggest that vulnerability in the thalamus may be determined by an inability to maintain spatial buffering of extracellular glutamate under energy-compromised conditions. Such an environment may be sufficient to set into motion event cascades in these cells that lead to the demise of the structure as a whole.

Diabetes is a chronic epidemic compounded by a burden of vascular complications including diabetic nephropathy. Diabetic nephropathy affects ~40% of patients and is characterised by increased urinary albumin excretion and decreased glomerular filtration rate. Diabetic patients exhibit ~75% decreased plasma thiamine concentration, linked to increased renal thiamine clearance. In streptozotocin-induced diabetic rats, decreased plasma thiamine concentration was also associated with a reduction in expression and activity of transketolase. Transketolase is a thiamine pyrophosphate-dependent enzyme and a critical component of the reductive pentose phosphate pathway, a metabolic pathway leading from glycolysis involved with the synthesis of ribose sugars. It is proposed that increasing the relative flux of glucose through the pentose phosphate pathway can ameliorate hyperglycaemic damage. This thesis investigates mechanisms mediating the increased renal thiamine clearance and the effects of thiamine therapy on type 2 diabetic patients with nephropathy. The hypothesis that hyperglycaemia increases flux through the hexosamine pathway, leading to increased O-glycosylation of the transcription factor Sp1 and subsequent decreased expression of thiamine transporters is investigated. Thiamine transporters in normal human kidney sections were found to be localised to the proximal tubule. Investigations in primary cultures of human proximal tubule epithelial cells and the HK-2 cell line have shown that there is a decreased expression (-48 to -80%) and abundance (-52 to -77%) of thiamine transporters in cells cultured in high glucose concentrations (26mM) with respect to low glucose concentrations (5 mM). There is only limited evidence supporting the involvement of the hexosamine pathway in these decreases. A double-blind, placebo-controlled study investigated the effect of thiamine supplementation on type 2 diabetic patients with microalbuminuria. Thiamine therapy restored plasma thiamine concentrations from 11nM to 98nM, exceeding the published median concentration observed in normal patients (64nM). After three months, thiamine therapy, but not placebo, caused a decrease in the urinary albumin excretion rate relative to baseline (-18 mg day-1). These results show promise for thiamine as a therapy for diabetic nephropathy.

Master of Science
Department of Grain Science and Industry
Greg Aldrich
Since 2010, there have been seven recalls related to thiamine deficiency in cat food products (FDA, 2017; FSA, 2017). Cats have a high requirement of thiamine (5.6 mg/kg), and deficiencies can lead to death within a month if not treated (AAFCO, 2017). A few studies have been published regarding the impact of retort processing on thiamine loss in canned pet food but no work has been reported on heat penetration in other containers (pouches and trays). Therefore, our objectives were to determine the effect of container size and type on thiamine retention during processing of cat food. Our hypothesis was that thiamine retention would be impacted by container size and type. To address this, a 2x3 factorial arrangement of treatments in which two container sizes (small: 89-104 mL vs medium: 163-207 mL) and three container types (can, pouch, and tray) were evaluated for B-vitamin losses and thermal process lethality of a wet pet food. A model wet cat loaf type formula was produced for all six experimental treatments and each was processed in duplicate over six-days. All ingredients including the vitamin premix (10x level) were thoroughly mixed, heated to 43ºC, and containers were manually filled. The filled and sealed containers were cooked in a retort (cans: SJ Reid Retort, Bellingham, WA; trays and pouches: FMC retort, Madera, CA) with thermocouples attached to the center of representative containers (n=14) in each batch. Software (Calsoft Systems, v. 5.0.5) was used to record the internal temperatures. The retort time was targeted to meet an F₀=8 at 121ºC and 21 PSI. Treatment sample were analyzed for included pH, moisture, crude protein, crude fat, ash, and B-vitamins. Results were analyzed using the GLM procedure in SAS (v. 9.4; Cary, NC) with means and interactions separated using Fisher LSD method by significant F and an α of 5%. The proximate composition and pH were similar (P > 0.10) among treatments. There was an interaction (P < 0.05) between container size and type for time to reach the F₀=8; wherein, the medium can and tray had the longest time (45.5 and 46.3 min, respectively); the small can and tray, and medium pouch were intermediate (35.4, 36.0, and 32.0 min, respectively); and the small pouch had the shortest time (36.0 min). There was no difference for either main effect of container type or size on heating lethality values (each main effect average F₀=10.3) and total lethality ranged from 12.7-16.7 min. Thiamine retention was lowest (70%) among the B-vitamins, and there was minimal loss throughout the process. The excess heating beyond F₀=8 may account for the dramatic impact on the retention of heat labile nutrients like thiamine. This may be more difficult to control in the newer packaging systems like pouches and trays.

Pyrithiamine-induced thiamine deficiency in rats is a well-established animal model of Wernicke's Encephalopathy (WE). This thesis project, submitted as four articles, presents an examination of metabolic events that contribute to the selective neuronal lesions observed in the medial thalamus (MT) of thiamine-deficient (TD) rat. In addition, the phenomenon of glucose-precipitated worsening of neurological status in WE patients (Wallis et al., 1978; Watson et al., 1981) is explored.
Lactate accumulation is known to occur selectively in regions of the TD brain, which ultimately express neuronal cell death (McCandless, 1982; Munujos et al., 1996). In Article 1, the metabolic origin and cellular localization of region-selective lactate accumulation in the MT of TD rats was studied using combined 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Parallel studies were performed to examine the effects of glucose loading on regional brain lactate synthesis in TD animals. Thiamine deficiency caused focal increases in the de novo synthesis of lactate via elevated glycolytic flux in the MT, while contribution via pyruvate recycling and the periphery remained nominal. Lactate levels remained unaltered in the frontal cortex (FC), a brain region that is spared in thiamine deficiency. Administration of a glucose load intensified the selective increases in lactate de novo synthesis and accumulation in the MT of TD rats, positing a role for lactic acidosis in the glucose-precipitated worsening of neurological status in TD patients. Accordingly, Article 2 addresses the effect of glucose loading on local cerebral pH in the vulnerable MT, compared to the FC, of TD rats. Administration of a glucose load resulted in detrimental decreases in regional pH selectively in the MT, implying that alterations of brain pH contribute to the pathogenesis of thalamic neuronal damage and consequent cerebral dysfunction in WE.
Region-specific alterations in the steady state levels of cerebral amino acid neurotransmitters have been well-documented in experimental animal models of thiamine deficiency (Butterworth et al., 1979; Butterworth & Heroux, 1989; Gaitonde et al., 1975; Plaitakis et al., 1979); however, the dynamics of these changes have never been systematically explored. In Article 3, we examined the metabolic fluxes through thiamine-dependent pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) using multinuclear NMR spectroscopy. Furthermore the cellular localization of the metabolic changes in relation to regional vulnerability to thiamine deficiency was addressed. Our studies clearly demonstrate that early decreases m metabolic flux through alpha-KGDH result in commensurate declines in aspartate concentrations in the MT of TD rats. Impairments to PDH flux manifest secondarily to the metabolic block at alpha-KGDH, likely due to depleted oxaloacetate pools. As a result of impaired pyruvate oxidation, declines in the de novo synthesis of glutamate and GABA ensue. The present findings also suggest that inhibition of flux through alpha-KGDH in TD brain occurs primarily in the neurons, while astrocytes possess compensatory mechanisms, i.e. the anaplerotic pathway, to replenish oxaloacetate concentrations via metabolic pathways that do not involve thiamine-dependent enzymes.
In Article 4, we investigated the regional effects of thiamine deficiency on the activity of thiamine-dependent branched-chain alpha-ketoacid dehydrogenase (BCKDH) and the resultant effects on regional cerebral branched-chain amino acid (BCAA) oxidation. Thiamine deficiency resulted in significant impairments in BCKDH activity; while parallel studies on enzyme distribution confirmed a lower oxidative capacity for BCAAs in the MT compared with the Fe.
The data presented in these four articles confirm and extend findings for the region-selective impairments in thiamine-dependent metabolic processes as the foundation of vulnerability of the MT to thiamine deficiency. In addition, glucose loading of TD rats exacerbates both lactic acidosis and impaired pyruvate oxidation in this vulnerable brain region, positing a role for these processes in the glucose-precipitated worsening of neurological status in TD patients. Impaired oxidative metabolism of glucose and BCAAs in the MT leads to the accumulation of potentially harmful metabolic intermediates, contributing to the mitochondrial dysfunction, cellular energy failure and ultimately neuronal cell death observed in thiamine deficiency.

Recruitment failure of lake trout (Salvelinus namaycush) in the Great Lakes has been attributed in part to the consumption of alewife (Alosa pseudoharengus) by adult lake trout, leading to Thiamine Deficiency Complex (TDC) and early mortality in fry. The current understanding of thiamine deficiency in lake trout fry is based on information from culture and hatchery settings, which do not represent conditions fry experience in the wild and may influence the occurrence of TDC. In the wild, lake trout fry have access to zooplankton immediately following hatching; previous studies found that wild fry begin feeding before complete yolk-sac absorption. However, hatchery-raised fry are not provided with food until after yolk-sac absorption, long after the development of TDC. Zooplankton are a potential source of dietary thiamine for wild fry in the early life stages that has not previously been considered in the occurrence of thiamine deficiency. We postulated that wild-hatched fry could mitigate thiamine deficiency through early feeding on natural prey. Specifically, we hypothesized 1) feeding should increase thiamine concentrations relative to unfed fry and 2) feeding should increase survival relative to unfed fry. Feeding experiments were conducted on lake trout fry reared from eggs collected from Lake Champlain in 2014 and Cayuga Lake in 2015. A fully crossed experimental design was used to determine the effect of early feeding by lake trout fry in thiamine replete and thiamine deplete treatments before and after feeding. Overall, thiamine concentrations and survival did not significantly differ between fed and unfed fry. Thiamine concentrations increased from egg stage to hatching in both years, suggesting a potential source of thiamine, which had not previously been considered, was available to the lake trout eggs during development.

Doctor of Philosophy (PhD)
Proteomics is rapidly achieving recognition as a complimentary and perhaps superior approach to examine global changes in protein abundance in complex biological systems and the value of these techniques in neuropsychiatry is beginning to be acknowledged. Characterizing the brain’s regional proteomes provides a foundation for the detection of proteins that may be involved in disease-related processes. Firstly, optimal conditions were achieved for the application of two dimensional-gel electrophoresis (2D-GE)-based proteomics with postmortem human brain tissue. These optimized techniques were then applied to soluble fractions of adjacent grey and white matter of a single cytoarchitecturally defined area (Brodmann area 9; BA9) and of two adjacent regions of frontal white matter (BA9 and CC body) from healthy individuals. These normative proteomic comparisons highlighted the importance of correct tissue sampling, i.e. proper separation of regional white matter, as heterogeneity in the respective proteomes was demonstrated. Furthermore, they stressed the necessity for future molecular brain mapping studies. The main focus of this thesis however, was to examine the proteomes of brain regions specifically vulnerable to alcohol-induced damage underlying cognitive dysfunction. Alcoholic patients commonly experience mild to severe cognitive decline. It is postulated that cognitive dysfunction is caused by an alcohol-induced region selective brain damage, particularly to the prefrontal cortex. The cerebellum is increasingly recognized for its role in various aspects of cognition and alcohol–induced damage to the cerebellar vermis could indirectly affect neurocognitive functions attributed to the frontal lobe. We used a 2D-GE-based proteomics approach to compare protein abundance profiles of BA9 grey and white matter and the cerebellar vermis from human alcoholics (neurologically uncomplicated and alcoholics complicated with liver cirrhosis) and healthy control brains. Among the protein level changes observed are disturbances in the levels of a number of thiamine-dependent enzymes. A derangement in energy metabolism perhaps related to thiamine deficiency seems to be important in all regions analysed, even where there are no clinical or pathological findings of Wernicke-Korsakoff Syndrome. Evidence of oxidative changes was also seen in all regions and effects of liver dysfunction in the vermis found. However, overall, these results highlight the complexity of this disease process in that a number of different proteins from different cellular pathways appear to be affected. By identifying changes in protein abundance levels in the prefrontal grey and white matter and the cerebellar vermis, hypotheses may draw upon more mechanistic explanations as to how chronic ethanol consumption causes the structural and functional alterations associated with alcohol-related brain damage. Furthermore, by comparing these results, we may be able to isolate disturbances in molecular pathways specific to the brain damage caused by alcohol, severe liver dysfunction and thiamine deficiency.

Proteomics is rapidly achieving recognition as a complimentary and perhaps superior approach to examine global changes in protein abundance in complex biological systems and the value of these techniques in neuropsychiatry is beginning to be acknowledged. Characterizing the brain’s regional proteomes provides a foundation for the detection of proteins that may be involved in disease-related processes. Firstly, optimal conditions were achieved for the application of two dimensional-gel electrophoresis (2D-GE)-based proteomics with postmortem human brain tissue. These optimized techniques were then applied to soluble fractions of adjacent grey and white matter of a single cytoarchitecturally defined area (Brodmann area 9; BA9) and of two adjacent regions of frontal white matter (BA9 and CC body) from healthy individuals. These normative proteomic comparisons highlighted the importance of correct tissue sampling, i.e. proper separation of regional white matter, as heterogeneity in the respective proteomes was demonstrated. Furthermore, they stressed the necessity for future molecular brain mapping studies. The main focus of this thesis however, was to examine the proteomes of brain regions specifically vulnerable to alcohol-induced damage underlying cognitive dysfunction. Alcoholic patients commonly experience mild to severe cognitive decline. It is postulated that cognitive dysfunction is caused by an alcohol-induced region selective brain damage, particularly to the prefrontal cortex. The cerebellum is increasingly recognized for its role in various aspects of cognition and alcohol–induced damage to the cerebellar vermis could indirectly affect neurocognitive functions attributed to the frontal lobe. We used a 2D-GE-based proteomics approach to compare protein abundance profiles of BA9 grey and white matter and the cerebellar vermis from human alcoholics (neurologically uncomplicated and alcoholics complicated with liver cirrhosis) and healthy control brains. Among the protein level changes observed are disturbances in the levels of a number of thiamine-dependent enzymes. A derangement in energy metabolism perhaps related to thiamine deficiency seems to be important in all regions analysed, even where there are no clinical or pathological findings of Wernicke-Korsakoff Syndrome. Evidence of oxidative changes was also seen in all regions and effects of liver dysfunction in the vermis found. However, overall, these results highlight the complexity of this disease process in that a number of different proteins from different cellular pathways appear to be affected. By identifying changes in protein abundance levels in the prefrontal grey and white matter and the cerebellar vermis, hypotheses may draw upon more mechanistic explanations as to how chronic ethanol consumption causes the structural and functional alterations associated with alcohol-related brain damage. Furthermore, by comparing these results, we may be able to isolate disturbances in molecular pathways specific to the brain damage caused by alcohol, severe liver dysfunction and thiamine deficiency.

Mild thiamin deficiency is prevalent in diabetes, and high dose thiamin ameliorates some diabetic complications, but there are no definitive studies addressing thiamin intake, diabetes control and thiamin status in diabetes. Subjects were 113 people with diabetes (58 type 1, 55 type 2), 43 with and 70 without thiamin supplementation. Dietary thiamin was estimated by 24-hour recall, diabetes control by HbA1c. Age, BMI, albumin excretion, activity level and smoking status did not correlate with red cell thiamin (RCT) in either group. RCT correlated with serum thiamin (ST) (p < 0.01). In those unsupplemented, adequate dietary thiamin did not ensure normal RCT, with 15.7 % of subjects below the reference range. Supplementation to intake > 4 mg/d, was significantly associated with normal RCT (p = 0.028), with 97.7% of supplemented subjects having normal RCT. Supplementation was also significantly associated with elevated serum thiamin 24 hours post supplementation, contrary to other reports. HbA1c was not significantly associated with RCT. Conclusions: In diabetes, adequate dietary thiamin does not ensure normal red cell thiamin, but supplementation to > 4 mg/day does, raising questions about actual thiamin requirements in diabetes and supporting evidence that thiamin deficiency in diabetes is not primarily due to dietary deficiency. Diabetes control was not significantly related to thiamin status.

Dissertação de Mestrado Integrado em Medicina Veterinária
RIAS, a Portuguese wildlife rehabilitation centre located in Algarve, has been admitting a substantial high number of seagulls, since its opening in October of 2009, with consistent clinical presentations pertaining to a paretic syndrome without cues of a particular disease. This preliminary study describes the clinical signs and microbiological, parasitological, toxicological and pathologic findings of paretic gulls received between 2009 and 2012. It tries to understand if there is an association between the manifestation of this disease and the different species and age classes affected. It seeks to determine possible relations between the geographic distribution of the cases and specific potentially problematic areas or human activities. All in order to additionally determine a probable cause for this disease taking into consideration the species affected, region where the animals were rescued and diseases that could explain the findings observed like: Newcastle disease, Salmonellosis, Aspergillosis, Sarcocystosis, Botulism, Algal toxicosis, Copper/Lead/Mercury intoxication, Organophosphorus/Carbamate poisoning and Thiamine deficiency. Additionally, a treatment trial with three therapeutic protocols (activated charcoal, fluid therapy and thiamine supplementation) was attempted to evaluate their influence in the outcome of the rehabilitation process and their value as a tentative diagnostic tools. Accordingly, digital records of 780 gulls were analyzed, as well as, results of more specific diagnostic ancillary tests used in carcasses and tissue samples in the centre and submitted to the Faculty of Veterinary Medicine of the University of Lisbon. From the 780 admissions, 148 gulls (18,97%) were found to have this paretic syndrome while alive, with L.fuscus and sub-adults being probably the classes most affected (p=0,02;p=0,00005). All these gulls, upon admission, were thin and dehydrated and the most frequent clinical signs documented were: depressed mental status without loss of conscious (58,8%); diarrhoea (43,9%), flaccid cloacae (70,3%); generalized muscular weakness (48,6%), moderate muscular weakness (46,6%); posterior paresis (69,6%) and moderate paresis (71,6%). Approximately half of the 148 gulls died while in rehabilitation and gross necropsy findings of paretic gulls were also unspecific and overall inconsistent. However, a high number of these gulls including dead admissions had a thin-walled cloacae distended with diarrhoea and the intestines were also displaying compatible signs of inflammation: oedema, vascular congestion and fluid faeces (32/71). Evidences of opportunistic diseases or development of confounding ailments like probably Aspergillosis were also noted. The differences between the therapeutic protocols were irrelevant (p=0,7422) and could not diagnose this condition. No pathogenic agent (bacterial or parasitic) capable of causing this syndrome was identified in the carcasses submitted (n=9). The necropsy examination and histopathology lesions reported in the faculty were inconclusive as to the cause of the paresis. Lead and Copper levels, analyzed in 2 gulls, were below what is considered in the literature as indicative of toxic. Nevertheless, in one of the gulls submitted a liver sample was positive for the presence of an organophosphorus compound, which could be in accordance with the high association measured between the spatial distribution of the proportion of paretic cases and density of several crops per municipality (Rho>0,5;p<0,05). In this moment, the data here compiled and the results obtained are still insufficient to determine or exclude the diseases in discussion as causes of this syndrome. Inconsistent use of ancillary tests results, paucity in the knowledge of ethologic and ecologic features of these birds in this region, irregularities in the retrieval of sick birds and tourism are some of the factors that may be influencing these results and should be addressed in future investigations.
RESUMO - SÍNDROME PARÉSICO EM GAIVOTAS (LARIDAE) NO SUL DE PORTUGAL - RIAS, centro de recuperação de animais selvagens localizado no Algarve, desde a sua abertura em Outubro de 2009 tem recebido um número elevado de gaivotas com um quadro clinico consistente com paresia, sem causa conhecida. Este estudo preliminar descreve os sinais clínicos e achados microbiológicos, parasitológicos, toxicológicos e anatomo/histopatológicos de gaivotas com parésia recebidas entre 2009 e 2012. Tenta igualmente perceber se existe uma associação entre a manifestação desta doença e as diferentes classes de idade e espécies afectadas. Procura determinar relações possíveis entre a distribuição geográfica dos casos e áreas/actividades humanas especificas e potencialmente problemáticas na área em estudo. Tudo com o intuito adicional de descobrir a causa provável desta doença tendo em consideração as espécies afectadas, região onde foram resgatadas e doenças que poderiam explicar os achados reunidos: Doença de Newcastle;Salmonelose;Aspergilose;Sarcocistose;Botulismo;Fitotoxicose;Intoxicação por cobre, chumbo, mercúrio;Intoxicação por organofosforados/carbamatos e Deficiência em tiamina. Três protocolos terapêuticos (carvão activado, fluidoterapia e tiamina) foram igualmente testados para avaliar os respectivos efeitos no processo de reabilitação e o seu valor diagnóstico. Desta forma, foram analisados registos de 780 gaivotas em conjunto com resultados obtidos de métodos de diagnóstico auxiliar mais específicos de carcaças e amostras recolhidas e analisadas no centro ou enviadas para a Faculdade de Medicina Veterinária da Universidade de Lisboa. Das 780 admissões, 148 larídeos (18,97%) exibiam em vida este síndrome, sendo provavelmente as classes mais afectadas: L.fuscus e sub-adultos (p=0,02;p=0,00005). Todas as gaivotas afectadas encontravam-se magras e desidratadas, sendo os sinais clínicos mais frequentemente documentados: depressão do estado mental (58,8%); diarreia (43,9%), cloaca flácida (70,3%); fraqueza muscular generalizada (48,6%), fraqueza muscular moderada (46,6%); paresia dos posteriores (69,6%) e paresia moderada (71,6%). Aproximadamente metade destes animais morreu no decurso da reabilitação e as lesões encontradas em necrópsia foram igualmente inespecíficas e inconsistentes. Contundo, um elevado número destes animais, incluindo admissões de animais mortos, apresentavam recurrentemente cloacas com parede finas e distendidas por diarreia e os intestinos apresentavam também sinais compatíveis com inflamação (32/71). Achados de doenças oportunistas ou capazes de provocar sinais/lesões semelhantes foram também reportados (e.g. Aspergilose). As diferenças obtidas entre os diferentes protocolos foram consideradas irrelevantes (p=0,7422) e incapazes de diagnosticar esta doença. Nenhum agente patogénico (bacteriano ou parasita) capaz de causar parésia foi identificado nas carcaças enviadas (n=9) e resultados de análise anatomo-histopatologia das lesões encontradas foram inconclusivos quanto à causa deste síndrome. Níveis de chumbo e cobre, analizados em amostras de fígado de 2 animais, encontravam-se abaixo do que é considerado na literatura como indicativo de tóxico. No entanto, em uma amostra de fígado de uma das gaivotas enviadas foram detectados resíduos de um organofosforado,o que poderá ser concordante com a elevada associação medida entre a distribuição espacial da proporção de casos com parésia e a densidade de diversas culturas por município (Rho>0,5;p<0,05). Neste momento, toda a informação aqui compilada é ainda insuficiente para determinar ou excluir as doenças em discussão enquanto causas. O inconsistente uso de métodos de diagnóstico auxiliar, a escassez de informação relativa à etologia e ecologia destes animais nesta região, irregularidades na recolha de animais doentes e o turismo são alguns dos factores que podem estar a influenciar estes resultados e deverão ser tidos em conta no futuro.

Mild thiamin deficiency is prevalent in diabetes, and high dose thiamin ameliorates some diabetic complications, but there are no definitive studies addressing thiamin intake, diabetes control and thiamin status in diabetes. Subjects were 113 people with diabetes (58 type 1, 55 type 2), 43 with and 70 without thiamin supplementation. Dietary thiamin was estimated by 24-hour recall, diabetes control by HbA1c. Age, BMI, albumin excretion, activity level and smoking status did not correlate with red cell thiamin (RCT) in either group. RCT correlated with serum thiamin (ST) (p < 0.01). In those unsupplemented, adequate dietary thiamin did not ensure normal RCT, with 15.7 % of subjects below the reference range. Supplementation to intake > 4 mg/d, was significantly associated with normal RCT (p = 0.028), with 97.7% of supplemented subjects having normal RCT. Supplementation was also significantly associated with elevated serum thiamin 24 hours post supplementation, contrary to other reports. HbA1c was not significantly associated with RCT. Conclusions: In diabetes, adequate dietary thiamin does not ensure normal red cell thiamin, but supplementation to > 4 mg/day does, raising questions about actual thiamin requirements in diabetes and supporting evidence that thiamin deficiency in diabetes is not primarily due to dietary deficiency. Diabetes control was not significantly related to thiamin status.

L’excitotoxicité est un mécanisme physiopathologique majeur impliqué dans la pathogenèse de la déficience en thiamine (DT). Dans les régions cérébrales vulnérables à la DT, on observe une mort cellulaire induite par excitotoxicité dont l’origine semble être la conséquence d’une perturbation du métabolisme énergétique mitochondrial, d’une dépolarisation membranaire soutenue et d’une diminution de l’absorption du glutamate par les astrocytes suite à la diminution de l’expression des transporteurs EAAT1 et EAAT2. Il est clairement établi que le glutamate joue un rôle central dans l’excitotoxicité lors de la DT. Ainsi, la mise en évidence des mécanismes impliqués dans la diminution de l’expression des transporteurs du glutamate est essentielle à la compréhension de la physiopathologie de la DT. L’objectif de cette thèse consiste en l’étude de la régulation des transporteurs astrocytaires du glutamate et la mise au point de stratégies thérapeutiques ciblant la pathogenèse de l’excitotoxicité lors de l’encéphalopathie consécutive à la DT. Les principaux résultats de cette thèse démontrent des perturbations des transporteurs du glutamate à la fois dans des modèles animaux de DT et dans des astrocytes en culture soumis à une DT. La DT se caractérise par la perte du variant d’épissage GLT-1b codant pour un transporteur du glutamate dans le thalamus et le colliculus inférieur, les régions cérébrales affectées lors d’une DT, en l’absence de modification des niveaux d’ARNm. Ces résultats suggèrent une régulation post-transcriptionnelle de l’expression des transporteurs du glutamate en condition de DT. Les études basées sur l’utilisation d’inhibiteurs spécifiques des facteurs de transcription NFkB et de l’enzyme nucléaire poly(ADP)ribose polymérase-1 (PARP-1) démontrent que la régulation de l’expression du transporteur GLT-1 est sous le contrôle de voies de signalisation NFkB dépendantes de PARP-1. Cette étude démontre une augmentation de l’activation de PARP-1 et de NFkB dans les régions vulnérables chez le rat soumis à une DT et en culture d’astrocytes DT. L’inhibition pharmacologique du facteur de transcription NFkB par le PDTC induit une augmentation des niveaux d’expression de GLT-1, tandis que l’inhibition de PARP-1 par le DPQ conduit à l’inhibition de l’hyperactivation de NFkB observée lors de DT. L’ensemble de ces résultats met en évidence un nouveau mécanisme de régulation des transporteurs du glutamate par l’activation de PARP-1. L’accumulation de lactate est une caractéristique de la DT. Un traitement avec le milieu de culture d’astrocytes en condition de DT sur des cultures d’astrocytes naïfs induit une diminution de l’expression de GLT-1 ainsi qu’une inhibition de la capacité d’absorption du glutamate par les astrocytes naïfs. En revanche, l’administration de lactate exogène ne modifie pas le niveau d’expression protéique de GLT-1. Ainsi, des facteurs solubles autres que le lactate sont sécrétés par des astrocytes en condition de perturbation métabolique et peuvent potentiellement réguler l’activité des transporteurs du glutamate et contribuer à la pathogenèse du syncytium astroglial. En outre, la ceftriaxone, un antibiotique de la famille des β-lactamines, augmente de façon différentielle l’expression du variant-d’épissage GLT-1 dans le colliculus inférieur chez le rat DT et en culture d’astrocytes DT. Ces résultats suggèrent que la ceftriaxone peut constituer une avenue thérapeutique dans la régulation de l’activité des transporteurs du glutamate lors de DT. Pour conclure, la mort cellulaire d’origine excitotoxique lors de DT survient en conséquence d’une dysfonction mitochondriale associée à une perturbation du métabolisme énergétique cérébral. La modification de l’expression des transporteurs du gluatamate est sous le contrôle des voies de signalisation NFkB dépendantes du facteur PARP-1. De plus, l’inhibition métabolique et l’augmentation des sécrétions de lactate observées lors de DT peuvent également constituer un autre mécanisme physiopathologique expliquant la diminution d’expression des transporteurs de glutamate. Enfin, la ceftriaxone pourrait représenter une stratégie thérapeutique potentielle dans le traitement de la régulation de l’expression des transporteurs du glutamate et de la perte neuronale associés à l’excitotoxicité observée lors de DT.
Excitotoxicity has been implicated as a major pathophysiological mechanism in the pathogenesis of thiamine deficiency (TD). Excitotoxic-mediated cell death is localized in areas of focal vulnerability in TD and may occur as a consequence of impairment in mitochondrial energy metabolism, sustained cell membrane depolarization and decreased uptake of glutamate by astrocytes due to the loss of excitatory amino acid transporters, (EAAT1 and EAAT2). Over the years, a number of studies have identified glutamate as being a major contributor to excitotoxicity in the pathophysiology of TD. Thus, downregulation of astrocytic glutamate transporters resulting in excitotoxicity is a key feature of TD and understanding the regulation of these transporters is essential to understanding the pathophysiology of the disorder. The objective of the present thesis project was to examine the underlying basis of astrocytic glutamate transporter regulation during TD encephalopathy. Major findings of the studies presented in this thesis project provide evidence for glutamate transporter abnormalities in TD animal models and astrocyte cultures exposed to TD. TD results in the loss of the glutamate transporter splice variant-1b (GLT-1b) in vulnerable areas of brain, i.e. thalamus and inferior colliculus, with no significant alteration in the mRNA levels of the transporters, suggesting that glutamate transporter regulation under conditions of TD is a posttranscriptional event. Studies using a specific inhibitor of the transcription factor, Nuclear factor-kappa B (NF-κB) and a nuclear enzyme poly (ADP)ribose polymerase-1 (PARP-1) provided evidence for the regulation of GLT-1 by PARP-1 dependent NF-κB signalling pathways. The major findings of this study suggested an increase in the activation of PARP-1 and NF-κB molecule in the vulnerable areas of TD rat brain and TD astrocyte cultures. Pharmacological inhibition of NF-κB showed an increase in the levels of GLT-1, while inhibition of PARP-1 using a specific PARP-1 inhibitor, DPQ inhibited the increased activation of NF-κB that was observed during TD. Overall results of this finding provided evidence for a mechanism involving PARP-1 activation in the regulation of glutamate transporters. Given the increased lactate accumulation as a classical feature of TD, we studied the effect of soluble factors produced by astrocytes on glutamate transporter function. Treatment of naïve astrocyte cultures with TD conditioned media resulted in decreased levels of GLT-1 and inhibition of glutamate uptake capacity concomitant with a loss of mitochondrial membrane potential. Administration of exogenous lactic acid produced a similar reduction in glutamate uptake to that resulting from conditioned media. However, lactic acid treatment did not result in a change in GLT-1 protein levels. In addition, the pro-inflammatory cytokine TNF-α was shown to be increased in astrocytes treated with TD along with elevated levels of the phospho-IκB fragment, indicative of increased activation of NFκB. Inhibition of NFκB led to an amelioration of the decrease in GLT-1 that occurs in TD, along with recovery of glutamate uptake. Thus, soluble factors released from astrocytes under conditions of metabolic impairment such as lactate and TNF-α impairment appear to exert a regulatory influence on glutamate transporter function. Ceftriaxone, a β-lactam antibiotic, has the ability to differentially stimulate GLT-1b (splice-variant) expression in the inferior colliculus in TD rats and under in vitro conditions with TD astrocyte cultures. Thus, ceftriaxone may be a potential therapeutic strategy in the regulation of glutamate transporter function during TD. In summary, excitotoxic cell death in TD occurs as a consequence of mitochondrial dysfunction associated with cerebral energy impairment and abnormal glutamate transporter status. A major underlying mechanism for glutamate transporter abnormalities is mediated by PARP-1 dependent NF-κB signaling pathways. In addition, metabolic inhibition with substantial production of lactate and TNF-α may be perhaps another mechanism responsible for glutamate transporter downregulation in TD.

碩士
國立陽明大學
生物化學研究所
92
Vitamin B1 (Thiamine) plays a critical role in energy and oxidative metabolism, its deficiency can result in abnormal oxidative degradation of nutrients. By treatment of cells with the thiamine antagonist amprolium as a means of inducing thiamine deficiency, we showed that thiamine deficiency elicited apoptosis in cultured cortical neuronal cells. The apoptosis was accompanied by the activation of caspases 3, 8, and 9 and a two-fold increase of reactive oxygen species (ROS) was detected. Treatment of cells with ROS inhibitors, NAC and vitamin C, did not prevent the amprolium induced death of neuronal cells. Western blot analysis using antibodies specific for phosphorylated MAP kinases demonstrated that amprolium treatment led to a decrease in the level of phosphorylated JNK and ERK. Exposure of cells to the JNK inhibitor SP600125, but not ERK inhibitor PD98059 and U0126, resulted in severe neuron death, suggesting that the activity of JNK is important to the survival of cortex neurons. The expression of p53 and its Ser-15 phosphorylation form was increased; and RT-PCR analysis indicated that the level of THTR-1, a p53 target gene, was upregulated. Mechanisms underlying the deficiency of vitamin B1 induced apoptosis remain to be determined.

Many salmonid populations are supported through captive breeding programs in which hatchery production supplies fish for reintroduction or supplementation efforts. In Lake Champlain, Atlantic salmon (Salmo salar) are the subject of a reintroduction effort that is complicated by the occurrence of thiamine (vitamin B1) deficiency in adult salmon returning to spawn. This deficiency results in high offspring mortality rates that must be mitigated by hatchery interventions (reviewed in Chapter 1). I used an experimental transcriptomics approach coupled with survival analyses to assess genetic variation in thiamine deficiency outcomes (i.e., survival at the family level) and identified candidate genes that may comprise a putatively adaptive response to selection imposed by thiamine deficiency (Chapter 2). Using sequence data from this study, I next compared patterns of genetic variation in the Lake Champlain population against two other populations to identify signatures of selection associated with hatchery rearing environment and differences in life history strategies (Chapter 3). Finally, I surveyed salmonid populations for density-dependent effects of adult spawning density on per capita fitness and found that in many cases, hatchery releases can contribute to decreased individual fitness. Using genotype data for returning adults in multiple populations, I also tested for reductions in effective population size (Ne) associated with hatchery supplementation and describe how increasing hatchery contribution to a population decreases Ne (Chapter 4). Together, my results demonstrate the powerful influences of hatchery supplementation on salmonid populations and suggest that specific modifications to hatchery practices can limit negative impacts of captive breeding on population genetic and demographic characteristics.

La documentation scientifique fait état de la présence, chez l’adulte, de cellules souches et progénitrices neurales (CSPN) endogènes dans les zones sous-ventriculaire et sous-granulaire du cerveau ainsi que dans le gyrus denté de l’hippocampe. De plus, un postulat selon lequel il serait également possible de retrouver ce type de cellules dans la moelle épinière et le néocortex des mammifères adultes a été énoncé. L’encéphalopathie de Wernicke, un trouble neurologique grave toutefois réversible qui entraîne un dysfonctionnement, voire une défaillance du cerveau, est causée principalement par une carence importante en thiamine (CT). Des observations récentes laissent envisager que les facteurs en cause dans la prolifération et la différenciation des CSPN pourraient également jouer un rôle important lors d’un épisode de CT. L’hypothèse, selon laquelle l’identification de nouveaux métabolites entrant dans le mécanisme ou la séquence de réactions se soldant en une CT pourraient en faciliter la compréhension, a été émise au moyen d'une démarche en cours permettant d’établir le profil des modifications métaboliques qui surviennent en de telles situations. Cette approche a été utilisée pour constater les changements métaboliques survenus au niveau du foyer cérébral dans un modèle de rats déficients en thiamine (rats DT), particulièrement au niveau du thalamus et du colliculus inférieur (CI). La greffe de CSPN a quant à elle été envisagée afin d’apporter de nouvelles informations sur la participation des CSPN lors d’un épisode de CT et de déterminer les bénéfices thérapeutiques potentiels offerts par cette intervention. Les sujets de l’étude étaient répartis en quatre groupes expérimentaux : un premier groupe constitué de rats dont la CT était induite par la pyrithiamine (rats DTiP), un deuxième groupe constitué de rats-contrôles nourris ensemble (« pair-fed control rats » ou rats PFC) ainsi que deux groupes de rats ayant subi une greffe de CSPN, soit un groupe de rats DTiP greffés et un dernier groupe constitué de rats-contrôles (rats PFC) greffés. Les échantillons de foyers cérébraux (thalamus et CI) des quatre groupes de rats ont été prélevés et soumis à des analyses métabolomiques non ciblées ainsi qu’à une analyse visuelle par microscopie à balayage électronique (SEM). Une variété de métabolites-clés a été observée chez les groupes de rats déficients en thiamine (rats DTiP) en plus de plusieurs métabolites dont la documentation ne faisait pas mention. On a notamment constaté la présence d’acides biliaires, d’acide cynurénique et d’acide 1,9— diméthylurique dans le thalamus, alors que la présence de taurine et de carnosine a été observée dans le colliculus inférieur. L’étude a de plus démontré une possible implication des CSPN endogènes dans les foyers cérébraux du thalamus et du colliculus inférieur en identifiant les métabolites-clés ciblant les CSPN. Enfin, les analyses par SEM ont montré une amélioration notable des tissus à la suite de la greffe de CSPN. Ces constatations suggèrent que l’utilisation de CSPN pourrait s’avérer une avenue thérapeutique intéressante pour soulager la dégénérescence symptomatique liée à une grave carence en thiamine chez l’humain.
Endogenous neural-stem progenitor cells (NSPC) have been documented to be found in the subventricular and subgranular zones, the dentate gyrus, and suggestions of the possibility of these cells being found in the spinal cord and neocortex in adult mammalian brain have been postulated. Thiamine deficiency (TD) is the major cause of Wernicke's Encephalopathy, a reversible neurological disorder that results in cerebral dysfunction and impairment. Recent evidence suggests factors involved in neural NSPC proliferation and differentiation are involved during TD. By means of a current approach for profiling metabolic changes occurring in focal areas of the TD rat brain, specifically the thalamus and the inferior colliculus (IC), it was hypothesized that new metabolites that might offer a better understanding into the sequel and/or mechanism of TD could be identified. It was also considered that the use of NSPC transplantation could offer new information into the involvement of NSPC and potential therapeutic benefit in TD. Non-targeted metabolomics analysis, fluorescences microscopy, and scanning election microscopy (SEM) analysis visualization was performed on samples of the focal areas (thalamus and IC) of pyrithiamine induced TD rats (PTD), pair-fed controls (PFC) rats, and NSPC transplanted TD and PFC rats. Various key metabolites were identified in rats with TD, including previous undocumented metabolites such as bile acids, kynurenic acid, and 1,9-dimethyluric acid in the thalamus and taurine and carnosine in the IC. The study also demonstrated a possible involvement of endogenous NSPC in focal areas of the thalamus and IC identifying key metabolites targeting NSPC and showed tissue amelioration (observed through SEM) following NSPC transplantation. The findings suggested that NSPC could offer a therapeutic alternative to alleviate some of symptomatic degeneration of TD.

Thiamin is a required coenzyme in the production of energy to fuel myocardial contraction. Therefore, thiamin deficiency (TD) may contribute to myocardial weakness by limiting the available energy for myocyte contraction. Previous studies report a wide range for the prevalence of TD in patients with heart failure (HF) (3% to 91%). These trials are limited by their small sample size, indirect measurement of thiamin status, exclusion criteria, and their focus on hospitalized patients. Therefore, this study determined the prevalence of TD in a large (n=100) group of ambulatory patients with HF, using high performance liquid chromotography. The prevalence of TD ([thiamin pyrophosphate (TPP)] ≤ 180 nM/l erythrocytes) was found to be 7%. TD was not related to furosemide use, dietary thiamin intake, severity of the HF, or age. More investigation into the factors that may influence development of TD in ambulatory patients with HF is warranted.

Thiamin transport in Escherichia coli is a model system to establish the tolerance of derivatives for transport into the cell. Since little is known about what types of thiamin derivatives may be successfully taken into the cell through the transport system, a series of thiamin derivatives are synthesized. A thiamin amino analog is synthesized and tested to determine the use of the analog as an alternate source of thiamin for growth of an E. coli thiamin mutant. Formate, acetate, and benzoate thiamin esters are synthesized and tested as alternate sources for growth of an E. coli thiamin mutant. Thiamin esters or amides may provide a scaffold for attaching other small molecules of interest to be imported into the cell by thiamin transport system. Thiamin containing formate, acetate, and benzoate esters were synthesized and tested as alternative growth source for thiamin using an E. coli mutant strain incapable of synthesizing thiamin. All three synthesized ester thiamin forms gave a zone of growth determined by disk-assay study. Also, an amino thiamin is synthesized to determine uptake through thiamin transport system by growth study using an E. coli mutant incapable of synthesizing thiamin. The growth curves resulting show concentration-dependent growth in the absence of natural thiamin, indicating amino thiamin is taken up by thiamin transport system as an alternate source of thiamin for growth. More characterization of the thiamin transport system is desired in order to develop thiamin conjugates of interest such as a photoaffinity probe for isolating thiamin-utilizing enzymes.

Le syndrome de Wernicke-Korsakoff (SWK) est un désordre neuropsychiatrique causé par la déficience en thiamine (DT). Dans la DT expérimentale comme dans le SWK, on observe une mort neuronale et des hémorragies dans certaines régions précises du diencéphale et du tronc cérébral. Les lésions diencéphaliques du SWK sont particulièrement sévères et entraînent souvent des séquelles amnésiques permanentes. Le lien entre la dysfonction métabolique induite par la DT et la mort neuronale n’est pas connu. Des rapports précédents ont démontré que la perméabilité de la barrière hémato-encéphalique (BHE) était altérée et ce, précédant l’apparition du dommage neuronal, suggérant un rôle critique de la dysfonction vasculaire. Les jonctions serrées (JS) interendothéliales, la base anatomique de la BHE, constituent un réseau moléculaire incluant l’occludin et les zonula occludens (ZOs). Cette thèse démontre une perte d’expression et une altération de la morphologie de ces protéines en relation avec la dysfonction de la BHE dans le thalamus de souris déficientes en thiamine, fournissant une explication pour la présence d’hémorragies. Le stress oxydatif peut entraîner des dommages directs aux protéines des JS et interférer avec leurs mécanismes de régulation. De plus, l’oxyde nitrique (NO) peut induire la métalloprotéinase matricielle-9 (MMP-9) impliquée dans la dégradation de ces protéines. L’endothélium vasculaire cérébral (EVC) semble être une source importante de NO dans la DT, l’expression de l’oxyde nitrique synthase endothéliale (eNOS) étant sélectivement induite dans les régions vulnérables. Le NO peut réagir avec les espèces réactives oxygénées et former du peroxynitrite, entraînant un stress oxydatif/nitrosatif endothélial. Les résultats présentés démontrent que la délétion du gène de eNOS prévient le stress oxydatif/nitrosatif cérébrovasculaire, l’extravasation des immunoglobulins G (IgGs) et l’altération de l’occludin et des ZOs dans le thalamus de souris déficientes en thiamine. De plus, cette délétion prévient l’induction de l’expression de MMP-9 dans l’EVC. Des résultats similaires ont été obtenus avec l’antioxydant N-acétylcystéine (NAC). Les mécanismes précis par lesquels les espèces réactives altèrent les protéines des JS sont inconnus. Caveolin-1, une composante majeure du caveolæ de l’EVC, est impliquée dans la régulation de l’expression des protéines des JS, et celle-ci est modulée par le stress oxydatif/nitrosatif; l’altération de l’expression de caveolin-1 a été récemment associée à la rupture de la BHE. Les résultats présentés démontrent que l’expression de caveolin-1 est sélectivement altérée dans l’EVC du thalamus de souris déficientes en thiamine, coïcidant avec la rupture de la BHE, et démontrent que la normalisation de l’expression de caveolin-1 par le NAC est associée avec l’atténuation du dommage à la BHE. Pris ensemble, ces résultats démontrent un rôle central du stress oxydatif/nitrosatif cérébrovasculaire, particulièrement celui provenant de eNOS, dans l’altération des JS de la BHE via des dommages directs et via l’induction de MMP-9 et de caveolin-1. Cette rupture de la BHE contribue par conséquent à la mort neuronale dans le thalamus, puisque la prévention des altérations cérébrovasculaires par la délétion du gène de eNOS et le NAC atténue significativement la mort neuronale. L’administration précoce d’antioxydants en combinaison avec la thiamine devrait donc être une considération importante pour le traitement du SWK.
Wernicke-Korsakoff syndrome (WKS) is a neuropsychiatric disorder caused by thiamine deficiency (TD). In experimental TD as in WKS, neuronal cell death and hemorrhages are observed in specific diencephalic and brainstem areas. Diencephalic lesions in WKS are especially severe and often lead to permanent amnesic symptoms. The link between TD-induced metabolic dysfunction and neuronal cell death is unknown. Previous reports have shown that blood-brain barrier (BBB) permeability was impaired and that this occurred prior to the onset of neuronal damage, suggesting a critical role for vascular dysfunction. Interendothelial tight junctions (TJs), the anatomical basis of the BBB, constitute a molecular network comprising occludin and zonula occludens (ZOs). This thesis shows a loss of expression and alterations in the morphology of these proteins in relation to BBB dysfunction in the thalamus of thiamine-deficient mice, providing an explanation for the presence of hemorrhages. Oxidative stress can lead to direct oxidative damage to TJ proteins and interfere with their regulation mechanisms. Also, nitric oxide (NO) can induce matrix metalloproteinase-9 (MMP-9) involved in the degradation of these proteins. Cerebral vascular endothelium (CVE) seems to be an important source of NO in TD, since endothelial nitric oxide synthase (eNOS) expression is selectively induced in vulnerable areas. NO can react with reactive oxygen species and form peroxynitrite, leading to endothelial oxidative/nitrosative stress. Results have show that eNOS gene deletion prevents cerebrovascular oxidative/nitrosative stress, immunoglobulins G (IgGs) extravasation and occludin and ZOs alterations in the thalamus of thiamine-deficient mice. Also, eNOS gene deletion prevents the induction of MMP-9 in CVE. Similar results have been obtained with the antioxidant N-acetylcysteine (NAC). Precise mechanisms by which reactive species alter TJ proteins are unknown. Caveolin-1, a major component of CVE caveolæ, is involved in the regulation of TJ protein expression, and is modulated by oxidative/nitrosative stress; alteration in caveolin-1 expression has been recently associated with BBB breakdown. The present results show that caveolin-1 expression is selectively altered in CVE of the thalamus of thiamine-deficient mice, and show that normalization of caveolin-1 expression by NAC is associated with the attenuation of BBB damage. Taken together, these results demonstrate a central role for cerebrovascular oxidative/nitrosative stress, especially coming from eNOS, in BBB TJ protein alterations via direct damage and via induction of MMP-9 and caveolin-1. As a result, BBB breakdown contributes to neuronal cell death in the thalamus, since prevention of cerebrovascular alterations by eNOS gene deletion and NAC significantly attenuates neuronal cell death. Early administration of antioxidants combined with thiamine should therefore be an important consideration for the treatment of WKS.