Thiamine Derivatives Research Articles

Identifying the Garlic and Grain Mixture with the Highest Allithiamine Content

Garlic (Allium sativum L.) has been extensively studied for its therapeutic and culinary applications, owing to its sulfur-containing bioactive compounds, including allicin and its derivatives. This study identified garlic varieties with high allicin content from different regions of Korea. It explores the synthesis of allithiamine, a lipid-soluble derivative of thiamine with enhanced bioavailability, by combining garlic with various grains. High-performance liquid chromatography (HPLC) analysis revealed significant regional variations in the allicin content, with Jeju garlic exhibiting the highest levels (1.04 mg/g). Among the grains tested, Avena sativa showed the most effective interaction with garlic, yielding the highest allithiamine levels (14.93 mg/g). These findings underscore the importance of grain matrix properties in optimizing the synthesis of allithiamine. This study provides valuable insights into the development of functional foods that leverage the bioactive compounds in garlic to enhance metabolic health and thiamine bioavailability.

Thiamine disulfide derivatives in thiol redox regulation: Role of thioredoxin and glutathione systems.

Thiamine (vitamin B1), under the proper conditions, is able to reversibly open the thiazole ring, forming a thiol-bearing molecule that can be further oxidized to the corresponding disulfide. To improve the bioavailability of the vitamin, several derivatives of thiamine in the thioester or disulfide form were developed and extensively studied over time, as apparent from the literature. We have examined three thiamine-derived disulfides: thiamine disulfide, sulbutiamine, and fursultiamine with reference to their intervention in modulating the thiol redox state. First, we observed that both glutathione and thioredoxin (Trx) systems were able to reduce the three disulfides. In particular, thioredoxin reductase (TrxR) reduced these disulfides either directly or in the presence of Trx. In Caco-2 cells, the thiamine disulfide derivatives did not modify the total thiol content, which, however, was significantly decreased by the concomitant inhibition of TrxR. When oxidative stress was induced by tert-butyl hydroperoxide, the thiamine disulfides exerted a protective effect, indicating that the thiol form deriving from the reduction of the disulfides might be the active species. Further, the thiamine disulfides examined were shown to increase the nuclear levels of the transcription factor nuclear factor erythroid 2 related factor 2 and to stimulate both expression and activity of NAD(P)H quinone dehydrogenase 1 and TrxR. However, other enzymes of the glutathione and Trx systems were scarcely affected. As the thiol redox balance plays a critical role in oxidative stress and inflammation, the information presented can be of interest for further research, considering the potential favorable effect exerted in the cell by many sulfur compounds, including the thiamine-derived disulfides.

Pre-analytical challenges from adsorptive losses associated with thiamine analysis

Thiamine (vitamin B1) is an essential vitamin serving in its diphosphate form as a cofactor for enzymes in the citric acid cycle and pentose-phosphate pathways. Its concentration reported in the pM and nM range in environmental and clinical analyses prompted our consideration of the components used in pre-analytical processing, including the selection of filters, filter apparatuses, and sample vials. The seemingly innocuous use of glass fiber filters, glass filter flasks, and glass vials, ubiquitous in laboratory analysis of clinical and environmental samples, led to marked thiamine losses. 19.3 nM thiamine was recovered from a 100 nM standard following storage in glass autosampler vials and only 1 nM of thiamine was obtained in the filtrate of a 100 nM thiamine stock passed through a borosilicate glass fiber filter. We further observed a significant shift towards phosphorylated derivatives of thiamine when an equimolar mixture of thiamine, thiamine monophosphate, and thiamine diphosphate was stored in glass (most notably non-silanized glass, where a reduction of 54% of the thiamine peak area was observed) versus polypropylene autosampler vials. The selective losses of thiamine could lead to errors in interpreting the distribution of phosphorylated species in samples. Further, some loss of phosphorylated thiamine derivatives selectively to amber glass vials was observed relative to other glass vials. Our results suggest the use of polymeric filters (including nylon and cellulose acetate) and storage container materials (including polycarbonate and polypropylene) for thiamine handling. Losses to cellulose nitrate and polyethersulfone filters were far less substantial than to glass fiber filters, but were still notable given the low concentrations expected in samples. Thiamine losses were negated when thiamine was stored diluted in trichloroacetic acid or as thiochrome formed in situ, both of which are common practices, but not ubiquitous, in thiamine sample preparation.

Abstract 1126: Thiamine Derivative Enhances Mitochondrial Function To Reduce Vascular Calcification

Introduction/Background: Vascular calcification significantly impacts cardiovascular disease and mortality. It's exacerbated by mitochondrial dysfunction in vascular smooth muscle cells (VSMCs), influenced by factors such as calcium and phosphate. This study examines the role of thiamine derivatives in improving mitochondrial function, potentially reducing vascular calcification. Research Question/Hypothesis: We hypothesize that thiamine derivatives enhance mitochondrial function, thereby attenuating vascular calcification. Goals/Aims: The aim is to assess the efficacy of thiamine derivatives in enhancing mitochondrial integrity and function to mitigate vascular calcification. Methods/Approach: VSMCs were treated in vitro with phosphate and calcium chloride to induce calcification, along with TTFD (Thiamine tetrahydrofurfuryl disulfide) treatment. Similarly, C57BL/6J mice were administered thiamine derivatives in vivo before inducing aortic calcification. Analysis included calcium deposition, apoptosis, and mitochondrial function assessment. Results/Data: TTFD significantly reduced vascular calcium deposition in VSMCs, both in vitro and in vivo. Mitochondrial function improvement was evidenced by increased oxygen consumption rates and JC-1 aggregate levels. Additionally, TTFD mitigated mitochondrial fission by inhibiting Drp1 phosphorylation at serine 616, leading to reduced apoptosis and osteogenic gene expression. Conclusion: Thiamine derivatives, particularly TTFD, demonstrate potential in mitigating vascular calcification through enhanced mitochondrial function and inhibition of mitochondrial fission. These findings highlight the therapeutic potential of thiamine derivatives in treating vascular calcification-related conditions.

Abstract 5385: Synthetic disulfide derivative of vitamin B1, fursultiamine, prevents VEGF-induced angiogenesis

Abstract Angiogenesis is a process in which new blood vessels are formed from the existing blood vessels required to supply nutrients and oxygen to various tissues. Angiogenesis plays a significant role in the growth and spread of many cancers, and several anti-angiogenic agents act as chemotherapeutic agents. Recent studies have shown that fursultiamine (thiamine tetrahydrofurfuryl disulfide), a lipid-soluble synthetic disulfide derivative of thiamine, possesses viable antineoplastic and antiviral effects. However, its role in preventing angiogenesis is not known. We hypothesize that fursultiamine, with its potent antioxidative actions, prevents VEGF-induced angiogenesis in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) were treated with VEGF in the absence and presence of fursultiamine (0-100 uM) in a time and dose-dependent manner. Cell viability was determined by MTT assay and in vitro angiogenesis by tube formation assays. The expression of various pro-angiogenic, anti-angiogenic and inflammatory markers will be determined by specific antibody arrays. Our results indicate that fursultiamine prevents VEGF-induced endothelial cell growth in a dose-dependent manner. Further, fursultiamine prevents the in vitro tube formation as determined tube formation assays. Further, fursultiamine also regulated the VEGF-induced expression of various angiogenic markers such as VEGF, PDGF, EGF, Ang1, Ang2, angiogenin, and FGF, inflammatory markers such IL-1b, GM-CSF, TGF-b, MMPs, and others such as TIMPs, PTX3 and CF-III. Based on our in vitro data, we suggest that fursultiamine could prevent VEGF-induced angiogenesis, and this compound as a potent anti-angiogenic agent that could prevent cancer growth and metastasis. Citation Format: Sabrina Tran, Zachary Frost, Kota Ramana. Synthetic disulfide derivative of vitamin B1, fursultiamine, prevents VEGF-induced angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5385.

Lipid-related thiamine deficiency cause mortality of river lampreys (Lampetra fluviatilis) during pre-spawning fasting

River lampreys (Lampetra fluviatilis) were caught in the fall 2014 on entering the River Perhonjoki for spawning and kept at a hatchery until spawning in late spring 2015 to produce larvae for compensatory stockings. Since the lampreys died massively from early February onwards, they were investigated in March and May to clarify the cause of the deaths. The symptoms in lampreys resembled those of lipid-related thiamine (vitamin B1) deficiency of salmonines, called the M74 syndrome in the Baltic Sea area. Because the lipid content of lampreys was known to be high, thiamine concentrations were analyzed in the liver and ovulated unfertilized eggs, and the mass, length, and whole-body lipid content were also measured. The hepatic total thiamine (TotTh) concentration was significantly negatively correlated with the body lipid content and fatness index (mass to length ratio) in both females and males. In females, the hepatic TotTh concentration was less than half that in males, and the most moribund lampreys were the largest and fattiest females. Females that survived until artificial stripping of the eggs were smaller, and their hepatic thiamine concentration was higher, and the fatness index lower, than in females in March. The concentration of free thiamine in the eggs had a stronger positive correlation with the hepatic TotTh concentration than the phosphorylated thiamine derivatives and was also significantly and negatively correlated with the fatness index and mass. It was concluded that increased lipid peroxidation due to the mobilization of polyunsaturated fatty acids from lipids in exogenous vitellogenesis consumed thiamine as an antioxidant during pre-spawning fasting—especially in the fattiest females—and thus caused their death. It is suggested that to ensure compensatory stockings, the largest lampreys could be thiamine-injected at the hatchery to improve their survival until stripping of the eggs and to improve the eggs’ thiamine status.

Wernicke-Korsakoff Syndrome in a Hunger Striker Despite Oral Thiamine Supplementation

CaseWe present the case of a 49-year-old woman who underwent a 237-day hunger strike on the streets of Pittsburgh, Pennsylvania. Despite medical supervision and appropriate micronutrient supplementation, including higher-than-recommended dosing of oral thiamine, the patient developed Wernicke’s Encephalopathy and subsequent Korsakoff Syndrome. She is now permanently impaired.IntroductionHunger strikers are subject to numerous sequelae of micronutrient deficiency. Among these are the paired conditions of Wernicke’s Encephalopathy and Korsakoff Syndrome, conditions secondary to prolonged thiamine deficiency. Unfortunately, few published guidelines exist regarding the medical management of hunger strikers. Preventative dosing guidance is borrowed from what is recommended for prolonged malnourishment from eating disorders or chronic alcohol use. Available guidelines are rarely academic. Rather, they were created by governmental agencies for the management of hunger strikers in prison or similar states of detention or incarceration. There is an indirect body of evidence that oral thiamine supplementation is rarely protective against the sequelae of thiamine deficiency in hunger strikers. We discuss supplementation recommendations and the evidence for their failure in practice. We briefly explore the historical evidence for the anatomic and physiologic changes of prolonged starvation that potentially explain this treatment failure, and offer alternatives to standard supplementation.ConclusionThe current recommendations of the management of hunger strikers regarding the prevention of thiamine deficiency are inadequate, and rarely prevent the clinical sequelae. Alternate management strategies need to be both researched and empirically used, while that research is being carried out. Novel lipid-soluble thiamine derivatives have promise, but prophylactic intravenous/intramuscular thiamine should be explored as the current standard of care.

Therapeutic potential of vitamin B1 derivative benfotiamine from diabetes to COVID-19.

Benfotiamine (S-benzoylthiamine-O-monophosphate), a unique, lipid-soluble derivative of thiamine, is the most potent allithiamine found in roasted garlic, as well as in other herbs of the genus Allium. In addition to potent antioxidative properties, benfotiamine has also been shown to be a strong anti-inflammatory agent with therapeutic significance to several pathological complications. Specifically, over the past decade or so, benfotiamine has been shown to prevent not only various secondary diabetic complications but also several inflammatory complications such as uveitis and endotoxemia. Recent studies also demonstrate that this compound could be used to prevent the symptoms associated with various infectious diseases such as HIV and COVID-19. In this review article, the authors discuss the significance of benfotiamine in the prevention of various pathological complications.

Molecular Structural Features that Determine the Neurotropic Activity of Thiamine Derivatives

Molecular Structural Features that Determine the Neurotropic Activity of Thiamine Derivatives

Update on Thiamine Triphosphorylated Derivatives and Metabolizing Enzymatic Complexes.

While the cellular functions of the coenzyme thiamine (vitamin B1) diphosphate (ThDP) are well characterized, the triphosphorylated thiamine derivatives, thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), still represent an intriguing mystery. They are present, generally in small amounts, in nearly all organisms, bacteria, fungi, plants, and animals. The synthesis of ThTP seems to require ATP synthase by a mechanism similar to ATP synthesis. In E. coli, ThTP is synthesized during amino acid starvation, while in plants, its synthesis is dependent on photosynthetic processes. In E. coli, ThTP synthesis probably requires oxidation of pyruvate and may play a role at the interface between energy and amino acid metabolism. In animal cells, no mechanism of regulation is known. Cytosolic ThTP levels are controlled by a highly specific cytosolic thiamine triphosphatase (ThTPase), coded by thtpa, and belonging to the ubiquitous family of the triphosphate tunnel metalloenzymes (TTMs). While members of this protein family are found in nearly all living organisms, where they bind organic and inorganic triphosphates, ThTPase activity seems to be restricted to animals. In mammals, THTPA is ubiquitously expressed with probable post-transcriptional regulation. Much less is known about the recently discovered AThTP. In E. coli, AThTP is synthesized by a high molecular weight protein complex from ThDP and ATP or ADP in response to energy stress. A better understanding of these two thiamine derivatives will require the use of transgenic models.

Metabolite discovery through global annotation of untargeted metabolomics data.

Liquid chromatography-high resolution mass spectrometry (LC-MS)-based metabolomics aims to identify and quantitate all metabolites, but most LC-MS peaks remain unidentified. Here, we present a global network optimization approach, NetID, to annotate untargeted LC-MS metabolomics data. The approach aims to generate, for all experimentally observed ion peaks, annotations that match the measured masses, retention times, and (when available) MS/MS fragmentation patterns. Peaks are connected based on mass differences reflecting adducting, fragmentation, isotopes, or feasible biochemical transformations. Global optimization generates a single network linking most observed ion peaks, enhances peak assignment accuracy, and produces chemically-informative peak-peak relationships, including for peaks lacking MS/MS spectra. Applying this approach to yeast and mouse data, we identified five previously unrecognized metabolites (thiamine derivatives and N-glucosyl-taurine). Isotope tracer studies indicate active flux through these metabolites. Thus, NetID applies existing metabolomic knowledge and global optimization to substantially improve annotation coverage and accuracy in untargeted metabolomics datasets, facilitating metabolite discovery.

Median Nerve Neuritis after Infection by the SARS-CoV-2 Virus

Background: The spectrum of neurological complications from SARS-CoV-2 infection is under constant investigation. According to numerous publications, occurrence of neurological complications can be about 36.4%. There are no reports to date of the occurrence of bilateral median nerve neuritis in the wrist after SARS-CoV-2 infection. Case: A previously healthy 24 years old female was observed in March 2021 due to dysesthesias in both hands, associated with nocturnal discomfort and decreased grip strength. The symptoms started a month earlier with acroparesthesias. SARS-CoV-2 virus infection was in January 2021 and evolved favorably, with progressive remission in about 10 days. Prior to the infection she didn’t have any of the hand symptoms; denied diabetes or metabolic diseases. Negative tests for SARS-CoV-2 infection since February. April 2021, the patient returned with persistent dysesthesias in her hands. Normal EMG, however with Sensitive Conduction Speed (SCS) of 50 m/sec on the right wrist; on the left SCS 55 m/sec. Sensitive latency 2.8 on the right and 2.6 on the left. Amplitude 17 on the right and 24 on the left. MRI detected an increase in signal in PDFS sequences in both median nerves, in the carpal tunnel, with no detected variations in cross-sectional areas or masses. The treatment was a 30 days 150 mg benfotiamine in two daily doses. Discussion: There is a wide variation in the clinical presentation of SARS-CoV-2 infection, ranging from asymptomatic patients to death. Most frequent symptoms are fever, dry cough, sore throat, dyspnea, myalgia, fatigue, chills, diarrhea, chest pain and vomiting, and the most common complications pneumonia and acute respiratory distress syndrome. Neurological manifestations of viral infection can occur in about 36.4% and in 45.5% in severe disease. They include Guillán-Barré and Miller Fischer syndrome; however, these conditions usually occur in more severe cases of the disease, but this patient hadn’t the typical picture of ataxia, ophthalmoplegia and areflexia. Benfotiamine is a synthetic derivative of thiamine with a multifaceted therapeutic profile, with a therapeutic role especially in diabetic neuropathy. Conclusion: Knowledge and recognition of neurological manifestations of SARS-CoV-2 infection and early detection of secondary peripheral neuropathies result in improvement of patients' clinical results and development of ideal ways of treatment. Although only a small percentage of patients develop peripheral neuropathy, in a pandemic this can result in a major social and health impact. The reported case is the first case of bilateral secondary median nerve neuritis in the literature. Level of evidence 4.

The ThiL enzyme is a valid antibacterial target essential for both thiamine biosynthesis and salvage pathways in Pseudomonas aeruginosa

Thiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. Thiamine biosynthesis occurs in bacteria but not in humans, so the enzymes in this pathway are attractive targets for antibiotic development. Among these enzymes, thiamine monophosphate kinase (ThiL) catalyzes the final step of this pathway, phosphorylating thiamine monophosphate (TMP) to produce TPP. Here, we extensively investigated ThiL in Pseudomonas aeruginosa, a major pathogen responsible for hospital-acquired infections. We demonstrate that thiL deletion abolishes not only thiamine biosynthesis, but also thiamine salvage capability, and results in growth defects of the ΔthiL strain even in the presence of thiamine derivatives, except for TPP. Most importantly, the pathogenesis of the ΔthiL strain was markedly attenuated compared with wild-type cells, with lower inflammatory cytokine induction and 103–104-fold decreased bacterial load in an in vivo infection model where the intracellular TPP level was in the submicromolar range. To validate P. aeruginosa ThiL (PaThiL) as a drug target, we further characterized its biochemical properties, determining a Vmax of 4.0 ± 0.2 nmol·min-1 and KM values of 111 ± 8 and 8.0 ± 3.5 μM for ATP and TMP, respectively. These comprehensive biological and biochemical results indicate that PaThiL represents a potential drug target for the development of an augmented repertoire of antibiotics against P. aeruginosa.

Interplay Between Thiamine and p53/p21 Axes Affects Antiproliferative Action of Cisplatin in Lung Adenocarcinoma Cells by Changing Metabolism of 2-Oxoglutarate/Glutamate.

Thiamine (vitamin B1) is often deficient in oncopatients, particularly those undergoing chemotherapy. However, interaction between the thiamine deficiency and anticancer action of drugs has not been characterized. A major natural thiamine derivative, thiamine diphosphate (ThDP), is a coenzyme of central metabolism, also known to affect transcriptional activity of the master metabolic regulator and genome guardian p53. A direct transcriptional target of p53, p21, regulates cell cycle dynamics and DNA damage response. Our work focuses on dependence of the action of the DNA damaging anticancer drug cisplatin on metabolic regulation through p53/p21 axes and cellular thiamine status in human lung adenocarcinoma cells A549. These cells are used as a model of a hardly curable cancer, known to develop chemoresistance to platinum drugs, such as cisplatin. Compared to wild type (A549WT), a stable line with a 60% knockdown of p21 (A549p21–) is less sensitive to antiproliferative action of cisplatin. In contrast, in the thiamine-deficient medium, cisplatin impairs the viability of A549p21– cells more than that of A549WT cells. Analysis of the associated metabolic changes in the cells indicates that (i) p21 knockdown restricts the production of 2-oxoglutarate via glutamate oxidation, stimulating that within the tricarboxylic acid (TCA) cycle; (ii) cellular cisplatin sensitivity is associated with a 4-fold upregulation of glutamic-oxaloacetic transaminase (GOT2) by cisplatin; (iii) cellular cisplatin resistance is associated with a 2-fold upregulation of p53 by cisplatin. Correlation analysis of the p53 expression and enzymatic activities upon variations in cellular thiamine/ThDP levels indicates that p21 knockdown substitutes positive correlation of the p53 expression with the activity of 2-oxoglutarate dehydrogenase complex (OGDHC) for that with the activity of glutamate dehydrogenase (GDH). The knockdown also changes correlations of the levels of OGDHC, GDH and GOT2 with those of the malate and isocitrate dehydrogenases. Thus, a p53/p21-dependent change in partitioning of the glutamate conversion to 2-oxoglutarate through GOT2 or GDH, linked to NAD(P)-dependent metabolism of 2-oxoglutarate in affiliated pathways, adapts A549 cells to thiamine deficiency or cisplatin treatment. Cellular thiamine deficiency may interfere with antiproliferative action of cisplatin due to their common modulation of the p53/p21-dependent metabolic switch between the glutamate oxidation and transamination.

Allithiamine Exerts Therapeutic Effects on Sepsis by Modulating Metabolic Flux during Dendritic Cell Activation.

Recent studies have highlighted that early enhancement of the glycolytic pathway is a mode of maintaining the pro-inflammatory status of immune cells. Thiamine, a well-known co-activator of pyruvate dehydrogenase complex, a gatekeeping enzyme, shifts energy utilization of glucose from glycolysis to oxidative phosphorylation. Thus, we hypothesized that thiamine may modulate inflammation by alleviating metabolic shifts during immune cell activation. First, using allithiamine, which showed the most potent anti-inflammatory capacity among thiamine derivatives, we confirmed the inhibitory effects of allithiamine on the lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and maturation process in dendritic cells. We applied the LPS-induced sepsis model to examine whether allithiamine has a protective role in hyper-inflammatory status. We observed that allithiamine attenuated tissue damage and organ dysfunction during endotoxemia, even when the treatment was given after the early cytokine release. We assessed the changes in glucose metabolites during LPS-induced dendritic cell activation and found that allithiamine significantly inhibited glucose-driven citrate accumulation. We then examined the clinical implication of regulating metabolites during sepsis by performing a tail bleeding assay upon allithiamine treatment, which expands its capacity to hamper the coagulation process. Finally, we confirmed that the role of allithiamine in metabolic regulation is critical in exerting anti-inflammatory action by demonstrating its inhibitory effect upon mitochondrial citrate transporter activity. In conclusion, thiamine could be used as an alternative approach for controlling the immune response in patients with sepsis.

On the nature of thiamine triphosphate in Arabidopsis.

Vitamin B1 is a family of molecules, the most renowned member of which is diphosphorylated thiamine (TDP)—a coenzyme vital for the activity of key enzymes of energy metabolism. Triphosphorylated thiamine derivatives also exist within this family, specifically thiamine triphosphate (TTP) and adenosine thiamine triphosphate (ATTP). They have been investigated primarily in mammalian cells and are thought to act as metabolic messengers but have not received much attention in plants. In this study, we set out to examine for the presence of these triphosphorylated thiamine derivatives in Arabidopsis. We could find TTP in Arabidopsis under standard growth conditions, but we could not detect ATTP. Interestingly, TTP is found primarily in shoot tissue. Drivers of TTP synthesis are light intensity, the proton motive force, as well as TDP content. In plants, TTP accumulates in the organellar powerhouses, the plastids, and mitochondria. Furthermore, in contrast to other B1 vitamers, there are strong oscillations in tissue levels of TTP levels over diel periods peaking early during the light period. The elevation of TTP levels during the day appears to be coupled to a photosynthesis‐driven process. We propose that TTP may signify TDP sufficiency, particularly in the organellar powerhouses, and discuss our findings in relation to its role.

The ThiL enzyme is a valid antibacterial target essential for both thiamine biosynthesis and salvage pathways in Pseudomonas aeruginosa

Thiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. Thiamine biosynthesis occurs in bacteria but not in humans; therefore, the enzymes in this pathway are attractive targets for antibiotic development. Among these enzymes, thiamine monophosphate kinase (ThiL) catalyzes the final step of this pathway, phosphorylating thiamine monophosphate to produce TPP. Here, we extensively investigated ThiL in Pseudomonas aeruginosa, a major pathogen responsible for hospital-acquired infections. We demonstrate that thiL deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability and results in growth defects of the ΔthiL strain even in the presence of thiamine derivatives, except for TPP. Most importantly, the pathogenesis of the ΔthiL strain was markedly attenuated, compared with that of WT cells, with lower inflammatory cytokine induction and 103-104-fold decreased bacterial loads in an in vivo infection model in which the intracellular TPP level was in the submicromolar range. To validate P. aeruginosa ThiL (PaThiL) as a drug target, we further characterized its biochemical properties, determining a Vmax of 4.0 ± 0.2 nmol·min-1 and Km values of 111 ± 8 and 8.0 ± 3.5 μm for ATP and thiamine monophosphate, respectively. An in vitro small-molecule screening assay identified PaThiL inhibitors including WAY213613, a noncompetitive inhibitor with a Ki value of 13.4 ± 2.3 μm and potential antibacterial activity against P. aeruginosa These comprehensive biological and biochemical results indicate that PaThiL represents a potential drug target for the development of an augmented repertoire of antibiotics against P. aeruginosa.

Regulation of Thiamine (Vitamin B1)-Dependent Metabolism in Mammals by p53.

Transcriptional factor p53 is a master regulator of energy metabolism. Energy metabolism strongly depends on thiamine (vitamin B1) and/or its natural derivatives. Thiamine diphosphate (ThDP), which is a major thiamine derivative, affects p53 binding to DNA. In order to elucidate the mechanism of regulation of thiamine-dependent metabolism by p53, we assessed putative p53-binding sites near transcription starting points in genes coding for transporters and enzymes, whose function is associated with thiamine and/or its derivatives. The predictions were validated by studying cell metabolic response to the p53 inducer cisplatin. Expression of p53 and its known target, p21, has been evaluated in cisplatin-treated and control human lung adenocarcinoma A549 cells that possess functional p53 pathway. We also investigated the activity of enzymes involved in the thiamine-dependent energy metabolism. Along with upregulating the expression of p53 and p21, cisplatin affected the activities of metabolic enzymes, whose genes were predicted as carrying the p53-binding sites. The activity of glutamate dehydrogenase GDH2 isoenzyme strongly decreased, while the activities of NADP+-dependent isocitrate dehydrogenase (IDH) and malic enzymes, as well as the activity of 2-oxoglutarate dehydrogenase complex at its endogenous ThDP level, were elevated. Simultaneously, the activities of NAD+-dependent IDH, mitochondrial aspartate aminotransferase, and two malate dehydrogenase isoenzymes, whose genes were not predicted to have the p53-binding sequences near the transcription starting points, were upregulated by cisplatin. The p53-dependent regulation of the assayed metabolic enzymes correlated with induction of p21 by p53 rather than induction of p53 itself.

Thiamine Mono- and Diphosphate Phosphatases in Bovine Brain Synaptosomes

Neurodegenerative diseases are accompanied by changes in the activity of thiamine mono- and diphosphate phosphatases, but molecular identification of these mammalian enzymes is incomplete. In this work, the protein fraction of bovine brain synaptosomes displaying phosphatase activity toward thiamine derivatives was subjected to affinity chromatography on thiamine-Sepharose. Protein fractions eluted with thiamine (pH 7.4 or 5.6), NaCl, and urea were assayed for the phosphatase activity against thiamine monophosphate (ThMP), thiamine diphosphate (ThDP), and structurally similar purine nucleotides. Proteins in each fraction were identified by mass spectrometry using the SwissProt database for all organisms because of insufficient annotation of the bovine genome. Peptides of two annotated bacterial phosphatases, alkaline phosphatase L from the DING protein family and exopolyphosphatase, were identified in the acidic thiamine eluate. The abundance of peptides of alkaline phosphatase L and exopolyphosphatase in the eluted fractions correlated with ThMPase and ThDPase activities, respectively. The elution profiles of the ThMPase and ThDPase activities differed from the elution profiles of nucleotide phosphatases, thus indicating the specificity of these enzymes toward thiamine derivatives. The search for mammalian DING phosphatases in the eluates from thiamine-Sepharose revealed X-DING-CD4, mostly eluted by the acidic thiamine solution (pH 5.6). The identified exopolyphosphatase demonstrated structural similarity with apyrases possessing the ThDPase activity. The obtained results demonstrate that mammalian DING proteins and apyrases exhibit ThMPase and ThDPase activity, respectively.

Thiamine (vitamin B1) in septic shock: a targeted therapy.

Thiamine (vitamin B1) is a water-soluble vitamin essential for human health. Thiamine deficiency is causal and/or contributory in a number of debilitating diseases including beri-beri, the Wernicke-Korsakoff syndrome, optic neuropathy, and others. While thiamine deficiency is relatively rare in developed nations as a result of dietary supplementation, thiamine deficiency is more common in nutritionally compromised populations. Thiamine pyrophosphate, a thiamine derivative, is essential to the citric acid cycle and thiamine deficiency can result in impaired aerobic respiration and cellular energy production. Thiamine also plays an important role in the pentose phosphate pathway and other key metabolic processes. Although thiamine deficiency is a known cause of lactic acidosis, it has been recently evaluated as a potential contributor to refractory lactic acidosis and organ injury in septic shock and other shock states. In this article, we review the epidemiology of thiamine deficiency in septic shock and the existing evidence base supporting thiamine supplementation. We conclude that specific sepsis phenotypes may stand to benefit the most from thiamine supplementation, and efforts might be made to identify and supplement these patients early in their hospital course.