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.

In Vitro and In Silico Studies on Cytotoxic Properties of Oxythiamine and 2'-Methylthiamine.

It is important to search for cytostatic compounds in order to fight cancer. One of them could be 2'-methylthiamine, which is a thiamine antimetabolite with an additional methyl group at the C-2 carbon of thiazole. So far, the cytostatic potential of 2'-methylthiamine has not been studied. We have come forward with a simplified method of synthesis using commercially available substrates and presented a comparison of its effects, as boosted by oxythiamine, on normal skin fibroblasts and HeLa cancer cells, having adopted in vitro culture techniques. Oxythiamine has been found to inhibit the growth and metabolism of cancer cells significantly better than 2'-methylthiamine (GI50 36 and 107 µM, respectively), while 2'-methylthiamine is more selective for cancer cells than oxythiamine (SI = 180 and 153, respectively). Docking analyses have revealed that 2'-methylthiamine (ΔG -8.2 kcal/mol) demonstrates a better affinity with thiamine pyrophosphokinase than thiamine (ΔG -7.5 kcal/mol ) and oxythiamine (ΔG -7.0 kcal/mol), which includes 2'-methylthiamine as a potential cytostatic. Our results suggest that the limited effect of 2'-methylthiamine on HeLa arises from the related arduous transport as compared to oxythiamine. Given that 2'-methylthiamine may possibly inhibit thiamine pyrophosphokinase, it could once again be considered a potential cytostatic. Thus, research should be carried out in order to find the best way to improve the transport of 2'-methylthiamine into cells, which may trigger its cytostatic properties.

Design of thiamine analogues for inhibition of thiamine diphosphate (ThDP)-dependent enzymes: Systematic investigation through Scaffold-Hopping and C2-Functionalisation.

Thiamine diphosphate (ThDP), the bioactive form of vitamin B1, is an essential coenzyme needed for processes of cellular metabolism in all organisms. ThDP-dependent enzymes all require ThDP as a coenzyme for catalytic activity, although individual enzymes vary significantly in substrate preferences and biochemical reactions. A popular way to study the role of these enzymes through chemical inhibition is to use thiamine/ThDP analogues, which typically feature a neutral aromatic ring in place of the positively charged thiazolium ring of ThDP. While ThDP analogues have aided work in understanding the structural and mechanistic aspects of the enzyme family, at least two key questions regarding the ligand design strategy remain unresolved: 1) which is the best aromatic ring? and 2) how can we achieve selectivity towards a given ThDP-dependent enzyme? In this work, we synthesise derivatives of these analogues covering all central aromatic rings used in the past decade and make a head-to-head comparison of all the compounds as inhibitors of several ThDP-dependent enzymes. Thus, we establish the relationship between the nature of the central ring and the inhibitory profile of these ThDP-competitive enzyme inhibitors. We also demonstrate that introducing a C2-substituent onto the central ring to explore the unique substrate-binding pocket can further improve both potency and selectivity.

Oxidative Dearomatization of PLP in Thiamin Pyrimidine Biosynthesis in Candida albicans.

The yeast thiamin pyrimidine synthase THI5p catalyzes one of the most complex organic rearrangements found in primary metabolism. In this reaction, the active site His66 and PLP are converted to thiamin pyrimidine in the presence of Fe(II) and oxygen. The enzyme is a single-turnover enzyme. Here, we report the identification of an oxidatively dearomatized PLP intermediate. We utilize oxygen labeling studies, chemical-rescue-based partial reconstitution experiments, and chemical model studies to support this identification. In addition, we also identify and characterize three shunt products derived from the oxidatively dearomatized PLP.

New Role of Water in Transketolase Catalysis.

Transketolase catalyzes the interconversion of keto and aldo sugars. Its coenzyme is thiamine diphosphate. The binding of keto sugar with thiamine diphosphate is possible only after C2 deprotonation of its thiazole ring. It is believed that deprotonation occurs due to the direct transfer of a proton to the amino group of its aminopyrimidine ring. Using mass spectrometry, it is shown that a water molecule is directly involved in the deprotonation process. After the binding of thiamine diphosphate with transketolase and its subsequent cleavage, a thiamine diphosphate molecule is formed with a mass increased by one oxygen molecule. After fragmentation, a thiamine diphosphate molecule is formed with a mass reduced by one and two hydrogen atoms, that is, HO and H2O are split off. Based on these data, it is assumed that after the formation of holotransketolase, water is covalently bound to thiamine diphosphate, and carbanion is formed as a result of its elimination. This may be a common mechanism for other thiamine enzymes. The participation of a water molecule in the catalysis of the one-substrate transketolase reaction and a possible reason for the effect of the acceptor substrate on the affinity of the donor substrate for active sites are also shown.

Thiamine metabolism genes in diatoms are not regulated by thiamine despite the presence of predicted riboswitches.

Thiamine pyrophosphate (TPP), an essential co-factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for c. 20% of global primary production. They have been predicted to contain TPP aptamers in the 3'UTR of some thiamine metabolism-related genes, but little information is known about their function and regulation. We used bioinformatics, antimetabolite growth assays, RT-qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum. We found that thiamine-related genes with putative TPP aptamers are not responsive to supplementation with thiamine or its precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), and targeted mutation of the TPP aptamer in the THIC gene encoding HMP-P synthase does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtTHIC is essential for thiamine biosynthesis and another gene, PtSSSP, is necessary for thiamine uptake. Our results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance.

Mechanistic Studies on the Single-Turnover Yeast Thiamin Pyrimidine Synthase: Characterization of the Inactive Enzyme.

The eukaryotic thiamin pyrimidine synthase, THI5p, has been identified as a suicidal/single-turnover enzyme that catalyzes the conversion of its active site histidine and lysine-bound pyridoxal phosphate (PLP) to the thiamin pyrimidine (HMP-P). Here we identify the histidine and PLP fragments using bottom-up proteomics and LC-MS analysis. We also identify the active form of the iron cofactor and quantitate the oxygen requirement of the THI5p reaction. This information is integrated into a mechanistic proposal for this remarkable reaction.

[Identification Methods for Pseudo-crystalline Polymorphs of Thiamine Chloride Hydrochloride, Which Transform Easily Depending on the Handling Environment].

Three forms of pseudo-crystalline polymorph of thiamine chloride hydrochloride are dependent on hydration states. We investigated how the measurement environment affects the transition of the pseudo-crystalline polymorph, and aimed to establish a reliable method of identifying the forms clearly by IR spectrophotometry. We prepared three pseudo-crystalline forms and compared their IR spectra. In the IR spectra obtained by the potassium chloride (KCl) disk method, Form II was identified based on its characteristic absorption, but Forms I and III could not be distinguished clearly. Form I transformed to Form III after mixing with undried KCl powder, and Form III transformed to Form I by simply being left in the laboratory environment. These results suggested that the reversible transformation between Forms I and III occurred depending on the hydration status during the process of measurement, as measured by the shift in the absorption wavenumber of the primary alcohol stretching vibration. In addition, Forms I and III could not be distinguished clearly by the X-ray powder diffraction and their crystalline forms were similar plate crystals. However, in the IR spectra by the attenuated total reflection (ATR) method, the three forms could be identified based on each characteristic absorption. In summary, the ATR method does not require pretreatment for sample analysis, can be performed quickly, and is thus suitable to identify crystalline polymorph forms such as pseudo-crystalline polymorphs of thiamine chloride hydrochloride, which transform easily depending on the hydration status in a measurement environment.

Formation scheme and some properties of a thiamine-derived pigment, pyrizepine, formed through the Maillard reaction.

Recently, a yellow Maillard pigment named pyrizepine was identified from a heated solution containing thiamine and glucose. Here, we examined the formation scheme of this pigment and some biological properties. The mass spectrometry and nuclear magnetic resonance data of pyrizepine prepared from [6-13C] glucose showed that the carbon at 6-position of glucose was inserted at 2 different positions of pyrizepine. 5-(Aminomethyl)-2-methylpyridin-4-amine (AMPA), a degradation product of thiamine, was detected in the reaction solution. The pigment also formed in the solution containing AMPA in place of thiamine. These results showed that pyrizepine formed from AMPA and C4 fragments derived from glucose. Pyrizepine showed antioxidative activities in the superoxide dismutase, 2,2-diphenyl-1-picrylhydrazyl, and H-ORAC assays. The pigment did not show mutagenicity with the Ames test. A trace amount of the pigment was detected in a pan-fried ground pork sample added glucose using liquid chromatography-tandem mass spectrometry.

A novel panel of yeast assays for the assessment of thiamin and its biosynthetic intermediates in plant tissues.

Thiamin (or thiamine), known as vitamin B1, represents an indispensable component of human diets, being pivotal in energy metabolism. Thiamin research depends on adequate vitamin quantification in plant tissues. A recently developed quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is able to assess the level of thiamin, its phosphorylated entities and its biosynthetic intermediates in the model plant Arabidopsis thaliana, as well as in rice. However, their implementation requires expensive equipment and substantial technical expertise. Microbiological assays can be useful in deter-mining metabolite levels in plant material and provide an affordable alternative to MS-based analysis. Here, we evaluate, by comparison to the LC-MS/MS reference method, the potential of a carefully chosen panel of yeast assays to estimate levels of total vitamin B1, as well as its biosynthetic intermediates pyrimidine and thiazole in Arabidopsis samples. The examined panel of Saccharomyces cerevisiae mutants was, when implemented in microbiological assays, capable of correctly assigning a series of wild-type and thiamin biofortified Arabidopsis plant samples. The assays provide a readily applicable method allowing rapid screening of vitamin B1 (and its biosynthetic intermediates) content in plant material, which is particularly useful in metabolic engineering approaches and in germplasm screening across or within species.

Aroma enhancement in dry cured loins by the addition of nitrogen and sulfur precursors.

Dry cured loins containing nitrogen (proline and ornithine) and sulfur (thiamine) compounds as precursors of aroma compounds at two concentration levels were manufactured. The effect of precursor addition on the microbiology and chemical parameters of loins was studied together with the aroma study performed by olfactometry and Free Choice Profile sensory analyses. Addition of precursors did not affect the microbial and chemical parameters, while aroma was affected when precursors were added at the highest level. The dry loin aroma profile was mainly composed by compounds 3-methylbutanal, methional, ethyl 3-methylbutanoate, 3-methylbutanoic acid, 1-octen-3-ol, 2-acetyl-1-pyrroline and 2-acetylpyrrole that contribute to musty, cooked potatoes, fruity, cheesy, mushroom, roasted and meaty odor notes. Proline and ornithine supplementation modified the loins aroma profile producing toasted odors, while the effect of thiamine supplementation on the aroma was revealed by the presence of sulfur derived compounds (methional and 2-methyl-3-(methylthio)furan) that contribute to the "cured meat odor".

The Chemistry behind ThiC Rearrangement.

Thiamine (vitamin B1) is crucial for life and plays a central role in metabolism. It contains thiazole and pyrimidine moieties that are constructed independently and then assembled together to generate thiamine phosphate. The study of the thiazole moiety is relatively clear, but deciphering the mechanistic enzymology of thiamine pyrimidine is more difficult. This review aims to summarize the recent research progress on ThiC rearrangement, mainly including the mechanism, related enzymes, and genes involved in the rearrangement.

Capillary-based fluorescence microsensor with polyoxometalates as nanozyme for rapid and ultrasensitive detection of artemisinin.

A novel capillary-based fluorescence microsensor for artemisinin was developed with functional polyoxometalates (POMs) as nanozyme by a layer-by-layer self-assembly strategy. Vanadomolybdophosphoric heteropoly acid (H5PMo10V2O40, PMoV2) and tungstophosphoric heteropoly acid (Na5PW11O39Cu, PW11Cu) with high peroxidase-like activity were synthesized and immobilized on capillary to catalyze artemisinin/thiamine reaction and generate the amplified fluorescence signal. The wide linear range up to 13.0 µM with the low limit of detection of 0.03 µM (S/N = 3) was achieved for the determination of artemisinin by using the proposed POMs-microsensor. The method has been successfully used to detect artemisinin in human plasma and antimalarial drugs with satisfactory accuracy. This work developed a novel capillary fluorescence microsensor with functional POMs as nanozyme, which can serve as a promising candidate in fluorescence microanalysis.

Thiamine Protects Glioblastoma Cells against Glutamate Toxicity by Suppressing Oxidative/Endoplasmic Reticulum Stress.

Thiamine (vitamin B1), which is synthesized only in bacteria, fungi and plants and which humans should take with diet, participates in basic biochemical and physiological processes in a versatile way and its deficiency is associated with neurological problems accompanied by cognitive dysfunctions. The rat glioblastoma (C6) model was used, which was exposed to a limited environment and toxicity with glutamate. The cells were stressed by exposure to glutamate in the presence and absence of thiamine. The difference in cell proliferation was evaluated in the XTT assay. Oxidative stress (OS) markers malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) levels, as well as endoplasmic reticulum (ER) stress markers 78-kDa glucose-regulated protein (GRP78), activating transcription factor-4 (ATF-4), and C/EBP homologous protein (CHOP) levels, were measured with commercial kits. Apoptosis determined by flow cytometry was confirmed by 4',6-diamidino-2-phenylindole (DAPI) staining. At all concentrations, thiamine protects the cells and increased the viability against glutamate-induced toxicity. Thiamine also significantly decreased the levels of MDA, while increasing SOD and CAT levels. Moreover, thiamine reduced ER stress proteins' levels. Moreover, it lessened the apoptotic cell amount and enhanced the live-cell percentage in the flow cytometry and DAPI staining. As a result, thiamine may be beneficial nutritional support for individuals with a predisposition to neurodegenerative disorders due to its protective effect on glutamate cytotoxicity in glioblastoma cells by suppressing OS and ER stress.

Ronald C.D. Breslow (1931-2017): A career in review.

Reviewed herein are key research accomplishments of Professor Ronald Charles D. Breslow (1931-2017) throughout his more than 60 year research career. These accomplishments span a wide range of topics, most notably physical organic chemistry, medicinal chemistry, and bioorganic chemistry. These topics are reviewed, as are topics of molecular electronics and origin of chirality, which combine to make up the bulk of this review. Also reviewed briefly are Breslow's contributions to the broader chemistry profession, including his work for the American Chemical Society and his work promoting gender equity. Throughout the article, efforts are made to put Breslow's accomplishments in the context of other work being done at the time, as well as to include subsequent iterations and elaborations of the research.

The Changes in Antioxidant Activity of Selected Flavonoids and Caffeine Depending on the Dosage and Form of Thiamine.

Phenolic compounds and thiamine may serve as therapies against oxidative stress-related neurodegenerative diseases. However, it is important to note that these components show high instability under changing conditions. The study's aim was to determine the impact of the thiamine concentration (hydrochloride-TH and pyrophosphate-TP; in the range 0.02 to 20 mg/100 g on the indices of the chelating properties and reducing power, and free radicals scavenging indices of EGCG, EGC, ECG and caffeine added from 0.04 to 6.0 mg/100 g. Our research confirmed that higher concentrations of TH and TP can exhibit significant activity against the test antioxidant indices of all components. When above 5.0 mg/100 g of thiamine was used, the radical scavenging abilities of the compound decreased in the following order: EGCG > ECG > EGC > caffeine. The highest correlation was found for the concentration of thiamine pyrophosphate to 20.0 mg/100 g and EGCG. Knowledge of the impact of factors associated with the concentration of both EGCG, EGC, ECG or caffeine and thiamine on their activity could carry weight in regulating the quality supplemented foods, especially of nutrition support for people of all ages were oral, enteral tube feeding and parenteral nutrition).

Development of carbon dot-thiochrome-based sensing system for ratiometric fluorescence detection of D-penicillamine.

A simple and rapid ratiometric fluorescent sensing system for D-penicillamine (D-PA) determination is developed based on yellow carbon dots (Y-CDs) combined with thiochrome (oxVB1) for the first time. The oxidization of thiamine (VB1) can be catalyzed by Alkaline-hydrolyzed artemisinin (a-ART) to form oxVB1, which leads to the occurrence of fluorescence emission peak at 466 nm. Furthermore, the oxidation reaction between a-ART and VB1 could be inhibited by D-PA, and accompanied with the decrease of fluorescence at 466 nm. However, the fluorescence peak of Y-CDs as an internal reference at 566 nm was almost unchanged. The ratiometric signal changes contributed to a robust and sensitive D-PA sensing. Under the optimal condition, a good linear response for the D-PA detection was obtained in the ranges of 0.5-50 µM with a detection limit of 0.33 µM. In addition, Y-CDs and thiochrome-based sensing system was applied to D-PA determination in real samples and obtained acceptable results. We developed a new carbon dots/thiochrome fluorescent nanoprobe for ratiometric fluorescence sensing of D-penicillamine.

Non-clinical safety profile and pharmacodynamics of two formulations of the anti-sepsis drug candidate Rejuveinix (RJX).

Here, we demonstrate that the two distinct formulations of our anti-sepsis drug candidate Rejuveinix (RJX), have a very favorable safety profile in Wistar Albino rats at dose levels comparable to the projected clinical dose levels. 14-day treatment with RJX-P (RJX PPP.18.1051) or RJX-B (RJX-B200702-CLN) similarly elevated the day 15 tissue levels of the antioxidant enzyme superoxide dismutase (SOD) as well as ascorbic acid in both the lungs and liver in a dose-dependent fashion. The activity of SOD and ascorbic acid levels were significantly higher in tissues of RJX-P or RJX-B treated rats than vehicle-treated control rats (p < 0.0001). There was no statistically significant difference between tissue SOD activity or ascorbic acid levels of rats treated with RJX-P vs. rats treated with RJX-B (p > 0.05). The observed elevations of the SOD and ascorbic acid levels were transient and were no longer detectable on day 28 following a 14-day recovery period. These results demonstrate that RJX-P and RJX-B are bioequivalent relative to their pharmacodynamic effects on tissue SOD and ascorbic acid levels. Furthermore, both formulations showed profound protective activity in a mouse model of sepsis. In agreement with the PD evaluations in rats and their proposed mechanism of action, both RJX-P and RJX-B exhibited near-identical potent and dose-dependent anti-oxidant and anti-inflammatory activity in the LPS-GalN model of ARDS and multi-organ failure in mice.

Effect of high pressure impregnation on micronutrient transfer in rice.

Rice, consumed by most people across the world, serves as a great mode for carrying nutrients. The processed, starch-rich white rice is mostly devoid of nutrients. High-pressure processing (HPP) is a technology known to produce cold gelatinizing effects in starch. This work shows the application of HPP in fortification of two types of white rice through high pressure gelatinization. The rice grains were impregnated with vitamin B1, calcium, and zinc. HPP treatment was carried out at the temperatures of 50 °C and 70 °C for up to 20 min. The samples were analysed for thiamine using the fluorometric method and minerals using ICP-MS. Results showed that the transfer of nutrients increases with treatment temperature and time, producing high level of nutrient uptake. HPP-fortified rice also showed stability after storage of two months. The moderate-temperature HPP has a great potential to be used as a method to produce a ready-to-eat variety of rice.

Low-dose thiamine supplementation of lactating Cambodian mothers improves human milk thiamine concentrations: a randomized controlled trial.

BACKGROUND: Infantile beriberi-related mortality is still common in South and Southeast Asia. Interventions to increase maternal thiamine intakes, and thus human milk thiamine, are warranted; however, the required dose remains unknown. OBJECTIVES: We sought to estimate the dose at which additional maternal intake of oral thiamine no longer meaningfully increased milk thiamine concentrations in infants at 24 wk postpartum, and to investigate the impact of 4 thiamine supplementation doses on milk and blood thiamine status biomarkers. METHODS: In this double-blind, 4-parallel arm randomized controlled dose-response trial, healthy mothers were recruited in Kampong Thom, Cambodia. At 2 wk postpartum, women were randomly assigned to consume 1 capsule, containing 0, 1.2 (estimated average requirement), 2.4, or 10 mg of thiamine daily from 2 through 24 weeks postpartum. Human milk total thiamine concentrations were measured using HPLC. An Emax curve was plotted, which was estimated using a nonlinear least squares model in an intention-to-treat analysis. Linear mixed-effects models were used to test for differences between treatment groups. Maternal and infant blood thiamine biomarkers were also assessed. RESULTS: In total, each of 335 women was randomly assigned to1 of the following thiamine-dose groups: placebo (n = 83), 1.2 mg (n = 86), 2.4 mg (n = 81), and 10 mg (n = 85). The estimated dose required to reach 90% of the maximum average total thiamine concentration in human milk (191 µg/L) is 2.35 (95% CI: 0.58, 7.01) mg/d. The mean ± SD milk thiamine concentrations were significantly higher in all intervention groups (183 ± 91, 190 ± 105, and 206 ± 89 µg/L for 1.2, 2.4, and 10 mg, respectively) compared with the placebo group (153 ± 85 µg/L; P < 0.0001) and did not significantly differ from each other. CONCLUSIONS: A supplemental thiamine dose of 2.35 mg/d was required to achieve a milk total thiamine concentration of 191 µg/L. However, 1.2 mg/d for 22 wk was sufficient to increase milk thiamine concentrations to similar levels achieved by higher supplementation doses (2.4 and 10 mg/d), and comparable to those of healthy mothers in regions without beriberi. This trial was registered at clinicaltrials.gov as NCT03616288.