Thiamine Acquisition Strategies Impact Metabolism and Competition in the Gut Microbe Bacteroides thetaiotaomicron.

Thiamine (vitamin B1) is an essential cofactor for all organisms. Humans primarily acquire thiamine through their diet, and thiamine deficiencies have adverse neurological effects. However, the role gut microbes play in modulating thiamine availability is poorly understood, and little is known about how thiamine impacts the stability of microbial gut communities. To investigate thiamine's role in the gut, we utilized the model gut microbe Bacteroides thetaiotaomicron. Transcriptome sequencing (RNA-seq) revealed a global downregulation of thiamine and amino acid biosynthesis, glycolysis, and purine metabolism when thiamine was present. Using genetic mutants with thiamine biosynthesis and transport locus mutations, we determined both systems were critical for growth in thiamine-deficient medium. The defect in the double transport mutant suggests an uncharacterized feedback mechanism between thiamine transport and biosynthesis in B. thetaiotaomicron. Mutant phenotypes were recapitulated during pairwise competitions, reinforcing the importance of encoding versatile thiamine acquisition mechanisms when thiamine concentrations are variable. In addition, liquid chromatography-mass spectrometry (LC-MS) analyses corroborate that exogenous thiamine levels affect the internal thiamine pool of B. thetaiotaomicron. Furthermore, we computationally examined the ability of other gut microbes to acquire thiamine and identified lineage-specific differences in thiamine acquisition strategies. Among the Bacteroidetes, the capacities for both thiamine transport and biosynthesis are common. Together, these data show that thiamine acquisition mechanisms used by B. thetaiotaomicron not only are critical for its physiology and fitness but also provide the opportunity to model how other gut microbes may respond to the shifting availability of thiamine in the gut. IMPORTANCE Variation in the ability of gut microbes to transport, synthesize, and compete for vitamin B1 (thiamine) is expected to impact the structure and stability of the microbiota, and ultimately this variation may have both direct and indirect effects on human health. Our study identifies the diverse strategies employed by gut Bacteroidetes to acquire thiamine. We demonstrate how the presence or absence of thiamine biosynthesis or transport dramatically affects the abundance of B. thetaiotaomicron in a competitive environment. This study adds further evidence that altering the presence or concentrations of water-soluble vitamins such as thiamine may be an effective method for manipulating gut community composition. In turn, targeted thiamine delivery could be used therapeutically to alter dysbiotic communities linked to disease. Author Video: An author video summary of this article is available

Glucose-induced down regulation of thiamine transporters in the kidney proximal tubular epithelium produces thiamine insufficiency in diabetes

Increased renal clearance of thiamine (vitamin B1) occurs in experimental and clinical diabetes producing thiamine insufficiency mediated by impaired tubular re-uptake and linked to the development of diabetic nephropathy. We studied the mechanism of impaired renal re-uptake of thiamine in diabetes. Expression of thiamine transporter proteins THTR-1 and THTR-2 in normal human kidney sections examined by immunohistochemistry showed intense polarised staining of the apical, luminal membranes in proximal tubules for THTR-1 and THTR-2 of the cortex and uniform, diffuse staining throughout cells of the collecting duct for THTR-1 and THTR-2 of the medulla. Human primary proximal tubule epithelial cells were incubated with low and high glucose concentration, 5 and 26 mmol/l, respectively. In high glucose concentration there was decreased expression of THTR-1 and THTR-2 (transporter mRNA: −76% and −53% respectively, p<0.001; transporter protein −77% and −83% respectively, p<0.05), concomitant with decreased expression of transcription factor specificity protein-1. High glucose concentration also produced a 37% decrease in apical to basolateral transport of thiamine transport across cell monolayers. Intensification of glycemic control corrected increased fractional excretion of thiamine in experimental diabetes. We conclude that glucose-induced decreased expression of thiamine transporters in the tubular epithelium may mediate renal mishandling of thiamine in diabetes. This is a novel mechanism of thiamine insufficiency linked to diabetic nephropathy

Neurological, Psychiatric, and Biochemical Aspects of Thiamine Deficiency in Children and Adults.

Thiamine (vitamin B1) is an essential nutrient that serves as a cofactor for a number of enzymes, mostly with mitochondrial localization. Some thiamine-dependent enzymes are involved in energy metabolism and biosynthesis of nucleic acids whereas others are part of the antioxidant machinery. The brain is highly vulnerable to thiamine deficiency due to its heavy reliance on mitochondrial ATP production. This is more evident during rapid growth (i.e., perinatal periods and children) in which thiamine deficiency is commonly associated with either malnutrition or genetic defects. Thiamine deficiency contributes to a number of conditions spanning from mild neurological and psychiatric symptoms (confusion, reduced memory, and sleep disturbances) to severe encephalopathy, ataxia, congestive heart failure, muscle atrophy, and even death. This review discusses the current knowledge on thiamine deficiency and associated morbidity of neurological and psychiatric disorders, with special emphasis on the pediatric population, as well as the putative beneficial effect of thiamine supplementation in autism spectrum disorder (ASD) and other neurological conditions

Aspects of the quantitative separation and estimation of thiamine and its phosphate esters : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Methods for the separation and estimation of thiamine, thiamine monophosphate and thiamine diphosphate which would be applicable to biological extracts were investigated. Two methods for the estimation of thiamine were compared, the acid dye method and the thiochrome method. The thiochrome method was preferred as the acid dye method was more difficult to perform and some interference by certain amino acids was indicated. As both methods only estimate free thiamine, the optimum conditions for hydrolysis of thiamine phosphate esters by wheat germ acid phosphatase were also investigated. High phosphatase concentrations in the digestion mixture interfered with the extraction of thiochrome, by isobutanol, after oxidation of the free thiamine produced. Variation of the buffer in which the digestion was performed also affected the recoveries obtained. The inclusion of magnesium ions in the digestion mixture increased the activity of the enzyme so that it was possible to use an amount of phosphatase which was low enough to avoid interference with the extraction of thiochrome but which was sufficient to completely hydrolyse thiamine phosphate esters. The presence of magnesium ions also prevented the interference observed when formate rather than acetate buffers were used in the digestion mixture. A variety of separation techniques were investigated. Compared to paper and thin layer chromatography, high voltage paper electrophoresis (at 3kV in pH 3.5 buffer) gave the best and quickest separations. However only a 60% recovery was obtained after samples were eluted from the paper with 0.1M hydrochloric acid. Separation was achieved by elution of the thiochrome derivatives of thiamine, TMP and TDP from Sephadex G10 gel. Recoveries, estimated spectrophotometrically, indicated that this method could be used for the quantitative separation of thiamine and its phosphate esters. However since the method does not allow concentration of samples, it would be unsuitable for the estimation of biological extracts. Separation of thiamine and its esters using three ion exchange resins was also investigated. Partial separation of thiamine and its phosphate esters was obtained with Amberlite GC50 resin, the separation being determined by the form of the resin used. The hydrogen form of the resin allowed separation between TDP and thiamine-TMP while the sodium form separated thiamine from TMP-TDP. Neither form of the resin bound TDP firmly even when water was used as the eluent, so that separation of TDP and TTP would not be possible. Separation was attempted by eluting samples from Dowex 1-X8 resin with formate buffers of increasing ionic strength or pH. While the separation of thiamine, TMP and TDP appeared to be complete, by the elution profile, it was found that sample breakdown occurred. Electrophoresis of the eluted samples showed that the only peak which contained a single component was that corresponding to thiamine. Sample break-down was further indicated by a low recovery obtained when a sample containing only TDP was eluted. Identification of the peak contaminants was attempted using high voltage electrophoresis but proved difficult due to salt retardation affecting the positions of the peak components after electrophoresis. With Dowex 50 resin TDP and TMP were easily separated and eluted with ammonium acetate buffer of varying pH and ionic strength but the elution of thiamine required high pH or ionic strength solutions. Sample breakdown also appeared to occur on elution of samples from the resin. When TMP and TDP were eluted, separation appeared to be complete but a recovery of greater than 100% was obtained for TMP and both eluted compounds exhibited a progressive breakdown after elution. Sample breakdown was particularly notable when thiamine alone was eluted as 2 peaks were eluted and, after oxidation, yellow fluorescent material as well as the usual blue (characteristic of thiochrome) was observed. Characterisation of the yellow fluorescent compound(s) was attempted using electrophoresis, ultra-violet spectra and fluorescent spectra and it was found to be similar, but not identical, to thiamine

Severe acute axonal neuropathy following treatment with arsenic trioxide for acute promyelocytic leukemia: a case report

Peripheral neuropathy is a common complication of arsenic toxicity. Symptoms are usually mild and reversible following discontinuation of treatment. A more severe chronic sensorimotor polyneuropathy characterized by distal axonal-loss neuropathy can be seen in chronic arsenic exposure. The clinical course of arsenic neurotoxicity in patients with coexistence of thiamine deficiency is only anecdotally known but this association may potentially lead to severe consequences. We describe a case of acute irreversible axonal neuropathy in a patient with hidden thiamine deficiency who was treated with a short course of arsenic trioxide for acute promyelocytic leukemia. Thiamine replacement therapy and arsenic trioxide discontinuation were not followed by neurological recovery and severe polyneuropathy persisted at 12-month follow-up. Thiamine plasma levels should be measured in patients who are candidate to arsenic trioxide therapy. Prophylactic administration of vitamin B1 may be advisable. The appearance of polyneuropathy signs early during the administration of arsenic trioxide should prompt electrodiagnostic testing to rule out a pattern of axonal neuropathy which would need immediate discontinuation of arsenic trioxide

Glutamine Phosphoribosylpyrophosphate Amidotransferase-independent Phosphoribosyl Amine Synthesis from Ribose 5-Phosphate and Glutamine or Asparagine

Phosphoribosylamine (PRA) is the first intermediate in the common pathway to purines and thiamine and is generated in bacteria by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (EC 2.4.2.14) from PRPP and glutamine. Genetic data have indicated that multiple, non-PRPP amidotransferase mechanisms exist to generate PRA sufficient for thiamine but not purine synthesis. Here we describe the purification and identification of an activity (present in both Escherichia coli and Salmonella enterica) that synthesizes PRA from ribose 5-phosphate and glutamine/asparagine. A purification resulting in greater than a 625-fold increase in specific activity identified 8 candidate proteins. Of the candidates, overexpression of AphA (EC 3.1.3.2), a periplasmic class B nonspecific acid phosphatase, significantly increased activity in partially purified extracts. Native purification of AphA to >95% homogeneity determined that the periplasmic L-asparaginase II, AnsB (EC 3.5.1.1), co-purified with AphA and was also necessary for PRA formation. The potential physiological relevance of AphA and AnsB in contributing to thiamine biosynthesis in vivo is discussed

Absorption of thiamine and nicotinic acid in the rat intestine during fasting and immobilization stress

By perfusion of isolated sections of intestine with a solution containing thiamine at a concentration of 3.1 micromole, it was established that thiamine absorption in animals fasted for 72 hours decreased by 28 percent, whereas absorption increased by 12 percent in rats after 24 hour immobilization. After immobilization, absorption of label in the intestinal mucosa increased. Na K ATPase activity in the intestinal mucosa decreased by 10 percent during fasting, and it increased with immobilization of the animals. Activity of Na K ATPase in the intestinal mucosa cells determined the absorption rate of thiamine and nicotinic acid at the level of vitamin transport through the plasma membranes of the enterocytes

Organic cation transporter 1 (OCT1) modulates multiple cardiometabolic traits through effects on hepatic thiamine content.

A constellation of metabolic disorders, including obesity, dysregulated lipids, and elevations in blood glucose levels, has been associated with cardiovascular disease and diabetes. Analysis of data from recently published genome-wide association studies (GWAS) demonstrated that reduced-function polymorphisms in the organic cation transporter, OCT1 (SLC22A1), are significantly associated with higher total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride (TG) levels and an increased risk for type 2 diabetes mellitus, yet the mechanism linking OCT1 to these metabolic traits remains puzzling. Here, we show that OCT1, widely characterized as a drug transporter, plays a key role in modulating hepatic glucose and lipid metabolism, potentially by mediating thiamine (vitamin B1) uptake and hence its levels in the liver. Deletion of Oct1 in mice resulted in reduced activity of thiamine-dependent enzymes, including pyruvate dehydrogenase (PDH), which disrupted the hepatic glucose-fatty acid cycle and shifted the source of energy production from glucose to fatty acids, leading to a reduction in glucose utilization, increased gluconeogenesis, and altered lipid metabolism. In turn, these effects resulted in increased total body adiposity and systemic levels of glucose and lipids. Importantly, wild-type mice on thiamine deficient diets (TDs) exhibited impaired glucose metabolism that phenocopied Oct1 deficient mice. Collectively, our study reveals a critical role of hepatic thiamine deficiency through OCT1 deficiency in promoting the metabolic inflexibility that leads to the pathogenesis of cardiometabolic disease

Thiamine Diphosphate in Whole Blood, Thiamine and Thiamine Monophosphate in Breast-Milk in a Refugee Population

BACKGROUND: The provision of high doses of thiamine may prevent thiamine deficiency in the post-partum period of displaced persons. METHODOLOGY/PRINCIPAL FINDINGS: The study aimed to evaluate a supplementation regimen of thiamine mononitrate (100 mg daily) at the antenatal clinics in Maela refugee camp. Women were enrolled during antenatal care and followed after delivery. Samples were collected at 12 weeks post partum. Thiamine diphosphate (TDP) in whole blood and thiamine in breast-milk of 636 lactating women were measured. Thiamine in breast-milk consisted of thiamine monophosphate (TMP) in addition to thiamine, with a mean TMP to total thiamine ratio of 63%. Mean whole blood TDP (130 nmol/L) and total thiamine in breast-milk (755 nmol/L) were within the upper range reported for well-nourished women. The prevalence of women with low whole blood TDP (&lt;65 nmol/L) was 5% and with deficient breast-milk total thiamine (&lt;300 nmol/L) was 4%. Whole blood TDP predicted both breast-milk thiamine and TMP (R(2) = 0.36 and 0.10, p&lt;0.001). A ratio of TMP to total thiamine ≥63% was associated with a 7.5 and 4-fold higher risk of low whole blood TDP and deficient total breast-milk thiamine, respectively. Routine provision of daily 100 mg of thiamine mononitrate post-partum compared to the previous weekly 10 mg of thiamine hydrochloride resulted in significantly higher total thiamine in breast-milk. CONCLUSIONS/SIGNIFICANCE: Thiamine supplementation for lactating women in Maela refugee camp is effective and should be continued. TMP and its ratio to total thiamine in breast-milk, reported for the first time in this study, provided useful information on thiamine status and should be included in future studies of breast-milk thiamine

Severe thiamine deficiency in eastern Baltic cod (Gadus morhua)

The eastern Baltic cod (Gadus morhua) population has been decreasing in the Baltic Sea for at least 30 years. Condition indices of the Baltic cod have decreased, and previous studies have suggested that this might be due to overfishing, predation, lower dissolved oxygen or changes in salinity. However, numerous studies from the Baltic Sea have demonstrated an ongoing thiamine deficiency in several animal classes, both invertebrates and vertebrates. The thiamine status of the eastern Baltic cod was investigated to determine if thiamine deficiency might be a factor in ongoing population declines. Thiamine concentrations were determined by chemical analyses of thiamine, thiamine monophosphate and thiamine diphosphate (combined SumT) in the liver using high performance liquid chromatography. Biochemical analyses measured the activity of the thiamine diphosphate-dependent enzyme transketolase to determine the proportion of apoenzymes in both liver and brain tissue. These biochemical analyses showed that 77% of the cod were thiamine deficient in the liver, of which 13% had a severe thiamine deficiency (i.e. 25% transketolase enzymes lacked thiamine diphosphate). The brain tissue of 77% of the cod showed thiamine deficiency, of which 64% showed severe thiamine deficiency. The thiamine deficiency biomarkers were investigated to find correlations to different biological parameters, such as length, weight, otolith weight, age (annuli counting) and different organ weights. The results suggested that thiamine deficiency increased with age. The SumT concentration ranged between 2.4-24 nmol/g in the liver, where the specimens with heavier otoliths had lower values of SumT (P = 0.0031). Of the cod sampled, only 2% of the specimens had a Fulton's condition factor indicating a healthy specimen, and 49% had a condition factor below 0.8, indicating poor health status. These results, showing a severe thiamine deficiency in eastern Baltic cod from the only known area where spawning presently occurs for this species, are of grave concern