Active Form Of Vitamin B6 Research Articles

Purine limitation prevents the exogenous pyridoxal 5'-phosphate accumulation of Salmonella enterica yggS mutants.

Pyridoxal 5'-phosphate is the active form of vitamin B6 and is an essential cofactor in all domains of life. PLP can be synthesized de novo or salvaged from the environment from one of the six B6 vitamers. B6 vitamer levels appear to be tightly regulated, and alterations in their levels can have deleterious effects, most notably being the development of B6-dependent epilepsy in humans. YggS homologs are broadly conserved across multiple organisms and considered to be involved in maintaining B6 homeostasis, though no specific mechanism has been defined. The current study showed that the exogenous accumulation of PLP caused by a lack of YggS can be prevented by purine limitation. The demonstration that purine limitation impacts exogenous PLP accumulation separates one consequence of a yggS mutation for further study and contributes to continuing efforts to define the biochemical and physiological roles of the COG0325 family of proteins.

Exploring the factors influencing the ketoenamine-enolimine tautomeric equilibrium of pyridoxal 5′-phosphate in branched-chain aminotransferases

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, is a critical coenzyme for various enzymes. It generates a Schiff base with the substrate and exhibits ketoenamine and enolimine tautomeric forms due to intramolecular proton transfer. This study aims to ascertain the predominant tautomeric form of the PLP Schiff base in MtIlvE and analyze its influencing factors. Molecular dynamics simulations indicate that the ketoenamine tautomer has higher binding free energy than the enolimine tautomer. Density functional theory calculations suggest that, despite their ability to interconvert at a relatively low energy barrier, the ketoenamine tautomer is thermodynamically more stable. Factors affecting the keto-enol tautomeric equilibrium were investigated by constructing various QM-cluster models. Our results demonstrate that both the protonation of the pyridine nitrogen and the presence of Tyr209, which stabilizes the O3 anion, shift the tautomeric equilibrium toward the ketoenamine configuration. These findings provide a theoretical basis for investigating enzyme catalytic mechanisms.

Recent advances in pyridoxal phosphate-dependent enzymes and their applications

Pyridoxal phosphate (PLP), the active form of vitamin B6, is an important coenzyme in various enzyme-catalyzed reactions. PLP-dependent enzymes can catalyze a variety of chemical reactions, such as racemization, decarboxylation, β-addition, β-elimination, retro-aldol cleavage, transamination, and α-elimination. They are biologically synthesized a powerful tool for a variety of natural amino acids, non-natural amino acids and their related compounds. This article details the structural features and catalytic mechanisms of typical PLP-dependent enzymes such as ω-transaminase, lysine decarboxylase, threonine aldolase, and L-tyrosine phenol-lyase, and reviews the research progress in molecular modification and industrial applications of these enzymes. Finally, this article provides an outlook on the future development of PLP-dependent enzymes, including in vivo regeneration system and industrial applications of PLP cofactors, and discusses the tremendous potential of these enzymes in biocatalytic applications.

The Z isomer of pyridoxilidenerhodanine 5'-phosphate is an efficient inhibitor of human pyridoxine 5'-phosphate oxidase, a crucial enzyme in vitamin B6 salvage pathway and a potential chemotherapeutic target.

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, acts as a cofactor in many metabolic processes. In humans, PLP is produced in the reactions catalysed by pyridox(am)ine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PDXK). Both PNPO and PDXK are involved in cancer progression of many tumours. The silencing of PNPO and PDXK encoding genes determines a strong reduction in tumour size and neoplastic cell invasiveness in models of acute myeloid leukaemia (in the case of PDXK) and ovarian and breast cancer (in the case of PNPO). In the present work, we demonstrate that pyridoxilidenerhodanine 5'-phosphate (PLP-R), a PLP analogue that has been tested by other authors on malignant cell lines reporting a reduction in proliferation, inhibits PNPO in vitro following a mixed competitive and allosteric mechanism. We also show that the unphosphorylated precursor of this inhibitor (PL-R), which has more favourable pharmacokinetic properties according to our predictions, is phosphorylated by PDXK and therefore transformed into PLP-R. On this ground, we propose the prototype of a novel prodrug-drug system as a useful starting point for the development of new, potential, antineoplastic agents.

VITAMIN B2 AND ITS STATUS IN VEGETARIANS AND VEGANS

The main goal of this review was to briefly highlight the key points of today’s knowledge about the role and metabolism of vitamin B2 as well as to compare its status in vegans, vegetarians, and omnivores according to present data. Biologically active forms of water-soluble vitamin B2 (riboflavin) are represented by flavin mononucleotide (FMN) and flavin adenine diphosphate (FAD), which are coenzymes in the reactions of the electron transport chain and the pyruvate dehydrogenase complex. Riboflavin is also involved in the metabolism of fatty acids, the synthesis of cholesterol and steroid hormones. Riboflavin also participates in the folate cycle. Vitamin B2 is crucial for the metabolism of other vitamins. FMN is necessary for the for-mation of the active form of vitamin B6, while FAD is necessary for the synthesis of niacin. Severe clinical insufficiency of vitamin B2 is not common. Besides insufficient riboflavin consumption, hypothyroidism can also be a cause of vitamin B2 deficiency as thyroxine regulates the conversion of ribo-flavin to its active forms  FMN and FAD. Lack of riboflavin can cause tissue damage; especially of the epithelial, reproductive, and nervous system. The variety of its manifestations is related to the fact that riboflavin deficiency also disrupts the metabolism of vitamins B6, or PP. Similarly, riboflavin deficiency can also disrupt the folate cycle, increasing the level of homocysteine which damages vascular intima and leads to endothelial dysfunction and atherosclerosis as a result. Rapid urinary excretion of riboflavin makes it safe even if taken in a high dose, changing the urine colour into bright-yellow. This safety of riboflavin makes it unharmful to be used as an oral tracer for monitoring compliance in clinical research. Ultra-high doses of ribo-flavin (more than 400 mg/day) can cause diarrhoea or polyuria. The content of vitamin B2 in animal-derived products is higher than in plant-based ones. This also determines its lower intake in vegans and vegetarians compared to omnivores. While there is a shred of evidence that plant-based diets in-crease the production of bioavailable riboflavin by the gut microbiota, this does not appear to be sufficient to completely compensate for the vegetari-ans’/vegans’ reduced dietary intake of vitamin B2, as most of the researches point the fact that vegans and vegetarians are more prone to lack vita-min B2 than omnivores. At the same time, it should be noted that there exists no universally adopted optimal method for assessing the status of vit-amin B2 in the body. Further research and standardization of methods may ease to assess the prevalence and risks of riboflavin deficiency in various dietary groups

Reported Awareness and Use of Pyridoxal-5'-Phosphate Supplementation in Alanine Aminotransferase and Aspartate Aminotransferase Assay Reagents: A Survey by the College of American Pathologists Clinical Chemistry Committee.

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) assay reagents are available both with and without supplementation with pyridoxal-5'-phosphate (P5P; the active form of vitamin B6), a catalytic cofactor required for their enzymatic reactions. Nonsupplemented assays may miss ALT or AST elevations in patients with vitamin B6 deficiency. To assess awareness and adoption of ALT and AST reagents that are supplemented with P5P. A 4-question survey about ALT and AST reagent supplementation with P5P was included in the College of American Pathologists General Chemistry and Therapeutic Drugs (C program) proficiency testing 2023 B mailing. Overall, 38% (1651 of 4304) of responding laboratories reported using ALT and/or AST reagent supplemented with P5P. P5P supplementation was more common for nonacademic hospital/medical center laboratories (44%; 713 of 1629) relative to other settings. Of the laboratories that reported not using P5P-supplemented reagents, few (5%; 141 of 2611) cited plans to convert in the future. Despite the availability of P5P-supplemented reagents from several major assay manufacturers, the most common stated barrier for adoption was that the laboratory's reagent manufacturer does not provide P5P-supplemented reagents. There is a lack of awareness of the existence and benefits of P5P-supplemented ALT and AST reagents. There is a need for ALT and AST assay manufacturers to clarify and standardize the P5P status of ALT and AST reagents.

Holo-transcobalamin is not associated with the risk of all-cause mortality in the general population

Background & AimsHolo-Transcobalamin (holo-TC) is the biologically active form of vitamin B12, a vitamin essential in human metabolism. The association between vitamin B12 (total cobalamin) and mortality risk has been controversially reported, whereas the relation between holo-TC and survival is unknown. In a population-based sample (n=862, female share 42.8 %, median age 62.3 years), we related serum holo-TC to the risk of all-cause mortality. MethodsWe measured serum holo-TC by electro-chemiluminescence. Multivariable-adjusted Cox proportional hazards regression models were used to quantify the association between serum holo-TC and all-cause mortality. ResultsOver a median follow-up time of 10.9 years, n=99 individuals died. We did not find significant associations between serum holo-TC and the risk of all-cause mortality (HR: 1.00 [95% CI 0.97–1.03] per 5-point increment in holo-TC), neither in the overall sample, nor in subgroups stratified by sex, diabetes, or hypertension. ConclusionThe biologically active form of vitamin B12, holo-TC, is not related to the risk of all-cause mortality in a moderate-sized sample from the general population.

7,8-Dihydroxyflavone is a direct inhibitor of human and murine pyridoxal phosphatase

Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5’-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

7,8-Dihydroxyflavone is a direct inhibitor of human and murine pyridoxal phosphatase.

Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5'-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

PNPO-PLP axis senses prolonged hypoxia in macrophages by regulating lysosomal activity.

Oxygen is critical for all metazoan organisms on the earth and impacts various biological processes in physiological and pathological conditions. While oxygen-sensing systems inducing acute hypoxic responses, including the hypoxia-inducible factor pathway, have been identified, those operating in prolonged hypoxia remain to be elucidated. Here we show that pyridoxine 5'-phosphate oxidase (PNPO), which catalyses bioactivation of vitamin B6, serves as an oxygen sensor and regulates lysosomal activity in macrophages. Decreased PNPO activity under prolonged hypoxia reduced an active form of vitamin B6, pyridoxal 5'-phosphate (PLP), and inhibited lysosomal acidification, which in macrophages led to iron dysregulation, TET2 protein loss and delayed resolution of the inflammatory response. Among PLP-dependent metabolism, supersulfide synthesis was suppressed in prolonged hypoxia, resulting in the lysosomal inhibition and consequent proinflammatory phenotypes of macrophages. The PNPO-PLP axis creates a distinct layer of oxygen sensing that gradually shuts down PLP-dependent metabolism in response to prolonged oxygen deprivation.

Pyridoxal 5'-Phosphate Biosynthesis by Pyridox-(am)-ine 5'-Phosphate Oxidase: Species-Specific Features.

Enzymes reliant on pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, hold significant importance in both biology and medicine. They facilitate various biochemical reactions, particularly in amino acid and neurotransmitter metabolisms. Vitamin B6 is absorbed by organisms in its non-phosphorylated form and phosphorylated within cells via pyridoxal kinase (PLK) and pyridox-(am)-ine 5'-phosphate oxidase (PNPOx). The flavin mononucleotide-dependent PNPOx enzyme converts pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate into PLP. PNPOx is vital for both biosynthesis and salvage pathways in organisms producing B6 vitamers. However, for those depending on vitamin B6 as a nutrient, PNPOx participates only in the salvage pathway. Transferring the PLP produced via PNPOx to client apo-enzymes is indispensable for their catalytic function, proper folding and targeting of specific organelles. PNPOx activity deficiencies due to inborn errors lead to severe neurological pathologies, particularly neonatal epileptic encephalopathy. PNPOx maintains PLP homeostasis through highly regulated mechanisms, including structural alterations throughout the catalytic cycle and allosteric PLP binding, influencing substrate transformation at the active site. Elucidation at the molecular level of the mechanisms underlying PNPOx activity deficiencies is a requirement to develop personalized approaches to treat related disorders. Finally, despite shared features, the few PNPOx enzymes molecularly and functionally studied show species-specific regulatory properties that open the possibility of targeting it in pathogenic organisms.

Identification of the pyridoxal 5'-phosphate allosteric site in human pyridox(am)ine 5'-phosphate oxidase.

Adequate levels of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6 , and its proper distribution in the body are essential for human health. The PLP recycling pathway plays a crucial role in these processes and its defects cause severe neurological diseases. The enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), whose catalytic action yields PLP, is one of the key players in this pathway. Mutations in the gene encoding PNPO are responsible for a severe form of neonatal epilepsy. Recently, PNPO has also been described as a potential target for chemotherapeutic agents. Our laboratory has highlighted the crucial role of PNPO in the regulation of PLP levels in the cell, which occurs via a feedback inhibition mechanism of the enzyme, exerted by binding of PLP at an allosteric site. Through docking analyses and site-directed mutagenesis experiments, here we identified the allosteric PLP binding site of human PNPO. This site is located in the same protein region as the allosteric site we previously identified in the Escherichia coli enzyme homologue. However, the identity and arrangement of the amino acid residues involved in PLP binding are completely different and resemble those of the active site of PLP-dependent enzymes. The identification of the PLP allosteric site of human PNPO paves the way for the rational design of enzyme inhibitors as potential anti-cancer compounds.

Impact of Pyridoxine Supplement on Oxidative Stress in Type 2 Diabetic Patients

Background: The physiologically active form of vitamin B6 is pyridoxal 5-phosphate (PLP), which functions as a coenzyme in 150 enzymatic processes such amino acid, carbohydrate, and lipid metabolism and is essential for the production and/or breakdown of neurotransmitters. . It also acts as an antioxidant by quenching Reactive Oxygen Species (ROS) and counteracting the formation of Advanced Glycation End-Product (AGEs). PLP is recycled by mammals from B6 vitamins found in diet and has been linked to a number of clinically significant disorders. This study aim was to evaluate the impact of pyridoxine supplementation on oxidative stress status in type 2 diabetic patients. Method: This prospective controlled randomized open-labeled study of newly diagnosed T2DM patients. The study was conducted from November 2022 to February 2023 at the Mesan Center for Diabetes and Endocrinology under the supervision of a specialist endocrinologist. The total number of participants whose data were collected in this study was one hundred and eight participants, eighty-eight patients newly diagnosed with type 2 diabetes were included in the study, and twenty of the participants were healthy subjects. The patients were allocated into three groups: Group 1: Control group, 20 T2DM patients were treated with non-pharmacological therapy (lifestyle modification) for one month, Group 2: 34 T2DM patients treated with metformin 500 mg/day in addition to non-pharmacological therapy (lifestyle modification) for one month, Group 3: 34 T2DM patients treated with metformin 500 mg/day plus vitamin B6 300 mg/day in addition to non-pharmacological therapy (lifestyle modification) for one month, in addition to Healthy subjects: 20 subjects were taken to compare the study parameters between type 2 diabetic patients and healthy persons at baseline. Measurement of Vitamin B6 (pyridoxine) blood level (PLP), body mass index (BMI) and Serum malondialdehyde level (MDA) was done in this study. Results: The results of the study showed that the use of pyridoxine supplementation plus metformin decreased the level of MDA. There was significant decrease in (MDA) level (P<0.01) pretreatment when compared to after treatment (3.85 μmole/L vs 1.66 μmole/L) respectively. Conclusion: Restoration of vitamin B6 level can leads to a decreased blood level of MDA in diabetic patients.

Discovery and characterization of natural product luteolin as an effective inhibitor of human pyridoxal kinase

Pyridoxal kinase (PDXK) is an essential enzyme in the synthesis of pyridoxal 5-phosphate (PLP), the active form of vitamin B6, which plays a pivotal role in maintaining the enzyme activity necessary for cell metabolism. Thus, PDXK has garnered attention as a potential target for metabolism regulation and tumor therapy. Despite this interest, existing PDXK inhibitors have faced limitations, including weak suppressive activity, unclear mechanisms of action, and associated toxic side effects. In this study, we present the discovery of a novel PDXK inhibitor, luteolin, through a high-throughput screening approach based on enzyme activity. Luteolin, a natural product, exhibits micromolar-level affinity for PDXK and effectively inhibits the enzyme's activity in vitro. Our crystal structures reveal that luteolin occupies the ATP binding pocket through hydrophobic interactions and a weak hydrogen bonding pattern, displaying reversible characteristics as confirmed by biochemical assays. Moreover, luteolin disrupts vitamin B6 metabolism by targeting PDXK, thereby inhibiting the proliferation of leukemia cells. This research introduces a novel screening method for identifying high-affinity and potent PDXK inhibitors and sheds light on clarification of the structural mechanism of PDXK-luteolin for subsequent structure optimization of inhibitors.

THU459 Hypophosphatasia With Normal ALPL Gene Test; A Case Report

Abstract Disclosure: K. Alkwatli: None. L.Z. Khan: None. Introduction: Hypophosphatasia is a rare hereditary disorder caused by loss of tissue nonspecific alkaline phosphatase activity, an essential enzyme in phosphate metabolism. Severe cases present perinatally and in early childhood. Mild cases can present in adulthood and can be misdiagnosed. We present an interesting patient who was clinically diagnosed with hypophosphatasia despite negative molecular genetic testing. Case presentation: 40 years-old female with a history of fibromyalgia and multiple bone fractures presented to the clinic with a long-standing history of multiple cavities, severe fatigue, headaches, muscle cramps, and arthralgia. The patient had numerous fractures throughout her life that started at age five without significant trauma, including wrist, forearm, and metatarsal bones. Her family history was negative. Laboratory testing showed low alkaline phosphatase levels on multiple occasions, 28,29,30 U/L (reference range 34-123). Her vitamin B6 was elevated at 309 nmol/L (20-125). Her zinc, calcium, vitamin D, and liver function tests were all within normal limits. Bone density scan showed a Z score of -1.0 at the lumbar spine and a Z score of -0.3 at the left femoral neck. Molecular genetic testing of the ALPL gene was negative. Hypophosphatasia was diagnosed based on her clinical and lab assessments, and she is being treated with Asfotase alfa. Her clinical course is yet to be determined. Discussion: ALPL gene is responsible for encoding the tissue nonspecific alkaline phosphatase (TNALP), the main enzyme affected in patients with hypophosphatasia. TNALP degrades inorganic pyrophosphate (PPi) to inorganic phosphate (Pi), an essential element for bone mineralization. It also facilitates vitamin B6 to cross the cell membranes. Therefore, loss of function mutations of the ALPL gene cause accumulation of PPi, which impairs the calcium-phosphate hemostasis and causes damage to multiple organs. In addition, the accumulation of the active form of vitamin B6 extracellularly can result in neurological damage.Patients present with a broad spectrum of musculoskeletal, renal, dental, and neurological symptoms. The variability of the phenotypic magnitudes is thought to be due to heterogeneity in the ALPL gene mutations. Over 400s of ALPL gene mutations have been identified in patients with hypophosphatasia. Novel variants are increasingly being reported.Negative genetic testing of the ALPL gene can still be seen in patients with hypophosphatasia due to either a variant that is undetectable by current testing methodology or if the causative gene/s are not on the ALPL gene panel. Conclusion: Genetic testing is helpful in confirming the disease and providing genetic counseling; however, treatment should still be considered if patients have sufficient clinical and laboratory findings. Presentation: Thursday, June 15, 2023

B-177 Quantitative Measurement of Vitamin B1 in Whole Blood using a Multiplex ESI-LC-MS/MS System

Abstract Background Vitamin B1 is a water-soluble vitamin required for human health. Thiamine diphosphate (TDP), the active form of vitamin B1, plays an essential role in biochemical pathways required for energy metabolism. TDP is analyzed routinely in the laboratory to assess the nutritional status of post-bariatric surgery patients and other individuals who are at risk of vitamin deficiency. In recent years, demand for vitamin B1 testing has increased dramatically, prompting development of a high throughput assay. Here, we present a method using protein precipitation and multiplex LC-ESI-MS/MS analysis at low pH with reversed-phase parameters that optimize chromatographic separation. The new method was validated for linearity, sensitivity, accuracy, imprecision, specificity, reproducibility, carryover, and stability. Method Whole blood samples were deproteinized using trichloroacetic acid (TCA) after the addition of stable-isotope-labeled TDP (d3-TDP) as an internal standard (IS). TDP was separated by reversed-phase chromatography on Waters XBridge™ Premier BEH C18 columns (2.1 × 100 mm, 2.5 µm) and analyzed on an Agilent StreamSelect™ LC-ESI-MS/MS platform. The StreamSelect™ system consists of four parallel Agilent Infinity Lab LC Series II instruments coupled to a single triple quadrupole mass spectrometer (6470B). TDP was found to interact with metal surfaces in the LC system, so the flow path was passivated using a deactivator solution which improved overall sensitivity and peak quality. TDP eluted in 3.15 ± 0.08 minutes; run times per column for a single stream and four streams were 9 min and 2.25 min, respectively. TDP was analyzed using positive electrospray ionization and multiple reaction monitoring mode with mass transitions of m/z 425.05 > 121.1 (quantifier) and m/z 425.05 > 304.00 (qualifier). Results The method was linear from 20 to 1000 nmol/L with an R2 > 0.99. Replicate analysis of low calibrators to evaluate assay sensitivity demonstrated accuracy within 10% and imprecision of ≤5%. Spike-recovery experiments showed an accuracy of ±15%. TDP measured in purchased, matrix-matched quality control (QC) and expired proficiency testing materials met the specified range for all samples. The intra- and inter-day assay imprecision was ≤3%. The cross-analyte interference study showed the analyte response in the IS-only blank was less than 10% of the LLOQ area. Also, the IS response at the ULOQ was ≤5% of the analyte area. Repeated injections of calibrators and QC materials across the four LC streams showed excellent parity (<2% imprecision). Re-injection reproducibility of extracted samples at 24 and 48 h was within ±2% of time zero concentration. Batch size tests indicated a high degree of robustness required to support the anticipated high testing volumes. Conclusions An accurate, specific, and high-throughput LC-ESI-MS/MS method was developed and validated to measure TDP in whole blood. Simple, quick sample preparation was employed for adaption to a high-throughput platform. StreamSelect™ using four parallel LCs reduced injection times to 2.25 min per sample and significantly minimized mass spectrometer idle time. The assay performance evaluations met our laboratory-developed test acceptance criteria for use in routine clinical laboratory analysis.

Fluorometric investigation of boric acid-pyridoxal 5′ phosphate interaction: A turn-on-fluorescence assay for the detection of boric acid in water bodies and fetal bovine serum

Boron is a trace element that is beneficial to plants as well as human and animal health. Boron supplementation is beneficial for absorbing vitamin D and magnesium, activating antioxidant enzymes and anticancer treatments. However, environmental accumulation of boron exceeding the permissible limit harms plants, animals and humans. Boric acid (BA), a form of boron, is known to interact with biomolecules such as NAD+/NADH, vitamin C, vitamin D, vitamin B1 and vitamin B6 determined through techniques like NMR, mass spectrometry, and FTIR. The fluorescence interaction of BA with the biologically active form of vitamin B6, Pyridoxal 5′ phosphate (PLP), has not been explored to date. Therefore, this study reports the interaction mechanism of BA with PLP via fluorescence spectroscopy analysis. The absorption, excitation and fluorescence lifetime measurements indicate that BA may form a complex through the mono or di-esterification of the phenolic hydroxyl group and aldehyde of PLP at different pH, forming the boronate esters. BA addition enhanced the fluorescence intensity of PLP by 12–22-fold in phosphate buffer (at acidic, neutral, and basic pH conditions). On the other hand, in citrate buffer at pH 4.0, BA enhanced PLP fluorescence by 28-fold. The binding constant for BA in phosphate buffer and citrate buffer was found to be 173 mol−1·L and 623 mol−1·L, respectively. At the same time, the detection limit for BA was found to be 445 μg/L and 212 μg/L in respective buffers. Therefore, PLP was used as a turn-on fluorescent probe to detect and quantify BA in water bodies and fetal bovine serum (FBS). The developed method successfully detected BA in environmental water bodies, achieving an average recovery rate of 99.84% (with a relative standard deviation (RSD) of 0.38%) in phosphate buffer and 81.59% (RSD, 4.56%) in citrate buffer. Furthermore, detecting BA in PLP-spiked FBS has been demonstrated in citrate buffer at pH 4.0, yielding an average recovery rate of 108.25% (RSD, 3.19%). Therefore, this study provides a basis for understanding the interaction of biomolecules like vitamins with boronates which aid in development of novel boronate based therapeutics and also helps in understanding the metabolic reactions of vitamin derivatives using fluorescence spectroscopy.

A synthetic cell-free 36-enzyme reaction system for vitamin B12 production

Adenosylcobalamin (AdoCbl), a biologically active form of vitamin B12 (coenzyme B12), is one of the most complex metal-containing natural compounds and an essential vitamin for animals. However, AdoCbl can only be de novo synthesized by prokaryotes, and its industrial manufacturing to date was limited to bacterial fermentation. Here, we report a method for the synthesis of AdoCbl based on a cell-free reaction system performing a cascade of catalytic reactions from 5-aminolevulinic acid (5-ALA), an inexpensive compound. More than 30 biocatalytic reactions are integrated and optimized to achieve the complete cell-free synthesis of AdoCbl, after overcoming feedback inhibition, the complicated detection, instability of intermediate products, as well as imbalance and competition of cofactors. In the end, this cell-free system produces 417.41 μg/L and 5.78 mg/L of AdoCbl using 5-ALA and the purified intermediate product hydrogenobyrate as substrates, respectively. The strategies of coordinating synthetic modules of complex cell-free system describe here will be generally useful for developing cell-free platforms to produce complex natural compounds with long and complicated biosynthetic pathways.

Bacterial lipopolysaccharide inhibits colonic carrier-mediated uptake of thiamin pyrophosphate: roles for TLR4 receptor and NF-κB/P38/JNK signaling pathway.

This study investigated the effect of the bacterial endotoxin lipopolysaccharide (LPS) on colonic uptake of thiamin pyrophosphate (TPP), the biologically active form of vitamin B1 that is generated by gut microbiota. We used three complementary models in our study: in vitro (human-derived colonic epithelial NCM460), ex vivo (human differentiated colonoid monolayers), and in vivo (mouse colonic tissue). The results showed that exposure of NCM460 cells to LPS leads to a significant inhibition of carrier-mediated TPP uptake as well as in decreased expression of the colonic TPP transporter (cTPPT) protein, mRNA, and heterologous nuclear RNA (hnRNA) compared with untreated controls. Similarly, exposure of human differentiated colonoid monolayers and mice to LPS caused significant inhibition in colonic carrier-mediated TPP uptake and in cTPPT protein, mRNA, and hnRNA expression. The effect of LPS on colonic TPP uptake and cTTPT expression was also found to be associated with a significant reduction in activity of the SLC44A4 promoter as well as in decreased expression of the nuclear factor Elf-3 (E74-like ETS transcription factor 3), which is needed for promoter activity. Finally, we found that knocking down the Toll-like receptor 4 (TLR4) and blocking the nuclear factor kappa B (NF-κB), JNK, and p38 signaling pathways with the use of pharmacological inhibitors lead to significant abrogation in the degree of LPS-mediated inhibition in TPP uptake and cTPPT expression. These results demonstrated that exposure of colonic epithelia to LPS inhibits colonic TPP uptake via transcriptional mechanism(s) and that the effect is mediated via TLR4 receptor and NF-κB/p38/JNK signaling pathways.NEW & NOTEWORTHY This study examined the effect of the bacterial lipopolysaccharide (LPS) on the colonic uptake of thiamin pyrophosphate (TPP), the biologically active form of vitamin B1. Three complementary models were used: in vitro (human NCM460 cells), ex vivo (human colonoids), and in vivo (mice). The results showed LPS to significantly suppress TPP uptake and the expression of its transporter, and that these effects are mediated via the membrane TLR4 receptor, and involve the NF-κB/p38/JNK signaling pathways.

Vitamin B6 regulates IL-33 homeostasis to alleviate type 2 inflammation.

Interleukin-33 (IL-33) is a crucial nuclear cytokine that induces the type 2 immune response and maintains immune homeostasis. The fine-tuned regulation of IL-33 in tissue cells is critical to control of the type 2 immune response in airway inflammation, but the mechanism is still unclear. Here, we found that healthy individuals had higher phosphate-pyridoxal (PLP, an active form of vitamin B6) concentrations in the serum than asthma patients. Lower serum PLP concentrations in asthma patients were strongly associated with worse lung function and inflammation. In a mouse model of lung inflammation, we revealed that PLP alleviated the type 2 immune response and that this inhibitory effect relied on the activity of IL-33. A mechanistic study showed that in vivo, pyridoxal (PL) needed to be converted into PLP, which inhibited the type 2 response by regulating IL-33 stability. In mice heterozygous for pyridoxal kinase (PDXK), the conversion of PL to PLP was limited, and IL-33 levels were increased in the lungs, aggravating type 2 inflammation. Furthermore, we found that the mouse double minute 2 homolog (MDM2) protein, an E3 ubiquitin-protein ligase, could ubiquitinate the N-terminus of IL-33 and sustain IL-33 stability in epithelial cells. PLP reduced MDM2-mediated IL-33 polyubiquitination and decreased the level of IL-33 through the proteasome pathway. In addition, inhalation of PLP alleviated asthma-related effects in mouse models. In summary, our data indicate that vitamin B6 regulates MDM2-mediated IL-33 stability to constrain the type 2 response, which might help develop a potential preventive and therapeutic agent for allergy-related diseases.