Biosynthesis of α-keto acids and resolution of chiral amino acids by l-amino acid deaminases from Proteus mirabilis.

Chiral amino acids and their deamination products, α-keto acids, have important applications in food, medicine, and fine chemicals. In this study, two l-amino acid deaminase genes from Proteus mirabilis, PM473 of type Ⅰ and PM471 of type Ⅱ were cloned and expressed in Escherichia coli respectively, expected to achieve the chiral separation of amino acids. Extensive substrate preference testing showed that both deaminases had catalytic effects on the d-amino acid component of the D, l-amino acids, and PM473 has a wider catalytic range for amino acids. When D, L-Cys was used as the substrate, all L-Cys components and 75.1 % of D-Cys were converted to mercapto pyruvate, and the remaining D-Cys was a single chiral enantiomer. Molecular docking analysis showed that the interaction between the substrate and the key residues affected the stereoselectivity of enzymes. The compatibility of hydrophobicity between the binding pocket and substrate may be the basic factor that affects the substrate selectivity. This work provides an alternative method for the production of α-keto acids and the resolution of chiral amino acids.

Transcriptomics and UHPLC-QQQ-MS analyses reveal the dysregulation of branched chain amino acids metabolism in renal fibrotic rats.

The dysregulated levels of branched chain amino acids (BCAA) contribute to renal fibrosis in chronic kidney disease (CKD), yet specific analysis of BCAA contents and how they are regulated still remain unclear. It is therefore of great scientific interest to understand BCAA catabolism in CKD and develop a sensitive method for simultaneous determination of individual BCAA and their metabolites branched chain α-ketoacids (BCKA). In this work, the important role of BCAA metabolism that drives renal fibrosis in the process of CKD was first revealed by using transcriptomics. The key target genes controlling BCAA metabolism were then validated, that is, mRNA levels of BCKDHA and BCKDHB, the regulating rate-limiting enzymes during BCAA metabolism were abnormally reduced by quantitative PCR (qPCR), and a similar drop-off trend of protein expression of BCKDH, HIBCH and MCCC2 that are closely related to BCAA metabolism was also confirmed by western blotting. Furthermore, we established a novel strategy that simultaneously determines 6 individual BCAA and BCKA in serum and tissue. The method based on dansylhydrazine derivatization and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-QQQ-MS) achieved to simultaneously determine the contents of BCAA and BCKA, which is efficient and stable. Compared with normal rats, levels of BCAA including leucine, isoleucine and valine in serum and kidney of CKD rats was decreased, while BCKA including α-ketoisocaproic acid, α-ketomethylvaleric acid and α-ketoisovaleric acid was increased. Together, these findings revealed the abnormality of BCAA metabolism in driving the course of kidney fibrosis and CKD. Our current study sheds new light on changes in BCAA metabolism during CKD, and may facilitate development of drugs to treat CKD and renal fibrosis.

Statin treatment reduces leucine turnover, but does not affect endogenous production of beta-hydroxy-beta-methylbutyrate (HMB).

BACKGROUND: Statins, or hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, are one of the most commonly prescribed medications for lowering cholesterol. Myopathic side-effects ranging from pain and soreness to critical rhabdomyolysis are commonly reported and often lead to discontinuation. The pathophysiological mechanism is, in general, ascribed to a downstream reduction of Coenzyme Q10 synthesis. HMG-CoA is a metabolite of leucine and its corresponding keto acid α-ketoisocaproic acid (KIC) and ß-hydroxy-ß-methylbutyrate (HMB), however, little is known about the changes in the metabolism of leucine and its metabolites in response to statins. OBJECTIVE: We aimed to investigate if statin treatment has implications on the upstream metabolism of leucine to KIC and HMB, as well as on other branched chain amino acids (BCAA). DESIGN: 12 hyperlipidemic older adults under statin treatment were recruited. The study was conducted as a paired prospective study. Included participants discontinued their statin treatment for 4 weeks before they returned for baseline measurements (before). Statin treatment was then reintroduced, and the participants returned for a second study day 7 days after reintroduction (after statin). On study days, participants were injected with stable isotope pulses for measurement of the whole-body production (WBP) of all BCAA (leucine, isoleucine and valine), along with their respective keto acids and HMB. RESULTS: We found a reduced leucine WBP (22 %, p = 0.0033), along with a reduction in valine WBP (13 %, p = 0.0224). All other WBP of BCAA and keto acids were unchanged. There were no changes in the WBP of HMB. CONCLUSIONS: Our study shows that statin inhibition of HMG-CoA reductase has an upstream impact on the turnover of leucine and valine. Whether this impairment in WBP of leucine may contribute to the known pathophysiological side effects of statins on muscle remains to be further investigated.

Dermonecrosis caused by a spitting cobra snakebite results from toxin potentiation and is prevented by the repurposed drug varespladib.

Snakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The venom of African spitting cobras often causes permanent injury via tissue-destructive dermonecrosis at the bite site, which is ineffectively treated by current antivenoms. To address this therapeutic gap, we identified the etiological venom toxins in Naja nigricollis venom responsible for causing local dermonecrosis. While cytotoxic three-finger toxins were primarily responsible for causing spitting cobra cytotoxicity in cultured keratinocytes, their potentiation by phospholipases A2 toxins was essential to cause dermonecrosis in vivo. This evidence of probable toxin synergism suggests that a single toxin-family inhibiting drug could prevent local envenoming. We show that local injection with the repurposed phospholipase A2-inhibiting drug varespladib significantly prevents local tissue damage caused by several spitting cobra venoms in murine models of envenoming. Our findings therefore provide a therapeutic strategy that may effectively prevent life-changing morbidity caused by snakebite in rural Africa.

A strategy to enhance the water solubility of luminescent ß-diketonate-Europium(III) complexes for time-gated luminescence bioassays.

Luminescent ß-diketonate-europium(III) complexes have been found a wide range of applications in time-gated luminescence (TGL) bioassays, but their poor water solubility is a main problem that limits their effective uses. In this work we propose a simple and general strategy to enhance the water solubility of luminescent ß-diketonate-europium(III) complexes that permits facile synthesis and purification. By introducing the fluorinated carboxylic acid group into the structures of ß-diketone ligands, two highly water-soluble and luminescent Eu3+ complexes, PBBHD-Eu3+ and CPBBHD-Eu3+, were designed and synthesized. An excellent solubility exceeding 20 mg/mL for PBBHD-Eu3+ was found in a pure aqueous buffer, while it also displayed strong and long-lived luminescence (quantum yield φ = 26%, lifetime τ = 0.49 ms). After the carboxyl groups of PBBHD-Eu3+ were activated, the PBBHD-Eu3+-labeled streptavidin-bovine serum albumin (SA-BSA) conjugate was prepared, and successfully used for the immunoassay of human α-fetoprotein (AFP) and the imaging of an environmental pathogen Giardia lamblia under TGL mode, which demonstrated the practicability of PBBHD-Eu3+ for highly sensitive TGL bioassays. The carboxyl groups of PBBHD can also be easily derivatized with other reactive chemical groups, which enables PBBHD-Eu3+ to meet diverse requirements of biolabeling technique, to provide new opportunities for developing functional europium(III) complex biolabels serving for TGL bioassays.

Relationship between Compound α-Ketoacid and Microinflammation in Patients with Chronic Kidney Disease.

Chronic kidney disease (CKD) refers to the presence of structural or functional abnormalities in the kidneys that affect health, lasting for more than 3 months. CKD is not only the direct cause of global incidence rate and mortality, but also an important risk factor for cardiovascular disease. Persistent microinflammatory state has been recognized as an important component of CKD, which can lead to renal fibrosis and loss of renal function, and plays a crucial role in the pathophysiology and progression of the disease. Simultaneously, compound α-Ketoacid can bind nitrogen-containing metabolites in the blood and accelerate their excretion from the body, thereby reducing the level of metabolic waste, alleviating gastrointestinal reactions in patients, and reducing the inflammatory response and oxidative stress state of the body. Compound α-Ketoacid contains amino acids required by CKD patients. In this review, we explore the relationship between compound α-Ketoacid and microinflammation in patients with CKD. The review indicated that compound α-Ketoacid can improve the microinflammatory state in CKD patients by improving the nutritional status of CKD patients, improving patient's acid-base balance disorder, regulating oxidative stress, improving gut microbiota, and regulating abnormal lipid metabolism.

Investigating the association between blood metabolites and telomere length: A mendelian randomization study.

BACKGROUND: Telomere length refers to the protective cap at the end of chromosomes, and it plays a crucial role in many diseases. The objective of this study is to explore the relationship between blood metabolites and telomere length, aiming to identify novel biological factors that influence telomere length. METHODS: In this study, we extracted genome-wide association study (GWAS) data for blood metabolites from a sample of 7824 Europeans. Additionally, GWAS data for telomere length were obtained from the Open GWAS database (GWAS ID: ieu-b-4879). The primary analysis of this study utilized the random inverse variance weighted (IVW) method. Complementary analyses were also conducted using the MR-Egger and weighted median approaches. Sensitivity analyses were performed to assess the robustness of the findings. These included the Cochran Q test, MR-Egger intercept test, MR-PRESSO, and leave-one-out analysis. To investigate the possibility of reverse causation, reverse MR analysis was conducted. Additionally, multivariable MR was utilized to evaluate the direct effect of metabolites on telomere length. RESULTS: The results suggested a potential association between 15-methylpalmitate, taurocholate, levulinate, and X-12712 and telomere length. MVMR analysis further showed that 15-methylpalmitate, taurocholate, and levulinate can directly influence telomere length, regardless of other metabolites. CONCLUSIONS: This study suggests that 15-methylpalmitate, taurocholate, and levulinate are likely factors correlated with telomere length. These findings will contribute to the development of strategies for protecting telomeres, preventing related diseases, and establishing a new biological foundation for achieving healthy aging.

Varespladib mitigates acute liver injury via suppression of excessive mitophagy on Naja atra envenomed mice by inhibiting PLA2.

Snakebite envenomation often leads to severe visceral injuries, including acute liver injury (ALI). However, the toxicity mechanism remains unclear. Moreover, varespladib can directly inhibit phospholipase A2 (PLA2) in snake venom, but its protective effect on snakebite-induced ALI and the mechanism have not been clarified. Previous studies have shown that snake venom PLA2 leads to neuron cell death via reactive oxygen species (ROS), one of the initial factors related to the mitophagy pathway. The present study group also found that ROS accumulation occurred after Naja atra envenoming. Hematoxylin and eosin (H/E) staining and immunohistochemistry (IHC) were performed to identify the expression of inflammatory factors in the liver tissue, and flow cytometry and immunofluorescence were used to detect ROS levels and mitochondrial function. Immunofluorescence and western blotting were also used for detecting mitophagy pathway-related proteins. The results showed that N. atra bite induced ALI by activating mitophagy and inducing inflammation and that varespladib had a protective effect. Collectively, these results showed the pathological mechanism of ALI caused by N. atra bite and revealed the protective effect of varespladib.

A Comparison of the Efficacy of Antivenoms and Varespladib against the In Vitro Pre-Synaptic Neurotoxicity of Thai and Javanese Russell's Viper (Daboia spp.) Venoms.

The heterogeneity in venom composition and potency in disparate Eastern Russell's viper (Daboia siamensis) populations has repercussions for the efficacy of antivenoms. This is particularly pronounced in geographical areas in which the venom of the local species has not been well studied and locally produced antivenoms are unavailable. In such cases, alternative therapies following envenoming, which are not limited by species specificity, may be employed to complement antivenoms. We studied the neuromuscular activity of D. siamensis venom from Thailand and Java (Indonesia) and the ability of Thai antivenoms and/or Varespladib to prevent or reverse these effects. Both Thai and Javanese D. siamensis venoms displayed potent pre-synaptic neurotoxicity but weak myotoxicity in the chick biventer cervicis nerve-muscle preparation. Whilst the neurotoxicity induced by both venoms was abolished by the prior administration of Thai D. siamensis monovalent antivenom or pre-incubation with Varespladib, Thai neuro-polyvalent antivenom only produced partial protection when added prior to venom. Pre-synaptic neurotoxicity was not reversed by the post-venom addition of either antivenom 30 or 60 min after either venom. Varespladib, when added 60 min after venom, prevented further inhibition of indirect twitches. However, the subsequent addition of additional concentrations of Varespladib did not result in further recovery from neurotoxicity. The combination of Thai monovalent antivenom and Varespladib, added 60 min after venom, resulted in additional recovery of twitches caused by either Thai or Javanese venoms compared with antivenom alone. In conclusion, we have shown that Varespladib can prevent and partially reverse the pre-synaptic neurotoxicity induced by either Thai or Javanese D. siamensis venoms. The efficacy of Thai D. siamensis monovalent antivenom in reversing pre-synaptic neurotoxicity was significantly enhanced by its co-administration with Varespladib. Further work is required to establish the efficacy of Varespladib as a primary or adjunct therapy in human envenoming.

Starch valorization: Direct conversion of starch to hexyl levulinate over SO4/ZrO2-KIT5 composite.

Valorization of biomass for the synthesis of valuable chemicals is a promising toolbox for replacing fossil fuel consumption. Long-chain hexyl levulinate (HL) is one of the attractive high-value chemicals obtained from biomass valorization. The current paper investigates the synthesis of KIT5-supported SO4/ZrO2 and its application in the successive hydrolysis and dehydration of starch to HL. The acidity of the prepared catalyst was modified, and its effect on the conversion of starch and HL yield was thoroughly studied. The parameters effective on the reaction yield and selectivity were optimized, and the possibility of 5-((hexyloxy)methyl) furan-2-carbaldehyde formation was explored. The prepared SO4/ZrO2-KIT5 can be used at least in four successive runs with a slight decrease in its reactivity. The HL yield was increased to a maximum of 28 %, while the starch conversion increased to a maximum of 100 % by conducting the reactions at 220 °C for 10 h. The accessibility and low cost of the starting materials as well as the method's simplicity, can give a practical outlook of its possible industrial application.

Lsr2 acts as a cyclic di-GMP receptor that promotes keto-mycolic acid synthesis and biofilm formation in mycobacteria.

Cyclic di-GMP (c-di-GMP) is a second messenger that promotes biofilm formation in several bacterial species, but the mechanisms are often unclear. Here, we report that c-di-GMP promotes biofilm formation in mycobacteria in a manner dependent on the nucleoid-associated protein Lsr2. We show that c-di-GMP specifically binds to Lsr2 at a ratio of 1:1. Lsr2 upregulates the expression of HadD, a (3R)-hydroxyacyl-ACP dehydratase, thus promoting the synthesis of keto-mycolic acid and biofilm formation. Thus, Lsr2 acts as a c-di-GMP receptor that links the second messenger's function to lipid synthesis and biofilm formation in mycobacteria.

Association of 2-oxoacid dehydrogenase complexes with respirasomes in mitochondria.

In the present study, cryo-electron tomography was used to investigate the localization of 2-oxoacid dehydrogenase complexes (OADCs) in cardiac mitochondria and mitochondrial inner membrane samples. Two classes of ordered OADC inner cores with different symmetries were distinguished and their quaternary structures modeled. One class corresponds to pyruvate dehydrogenase complexes and the other to dehydrogenase complexes of α-ketoglutarate and branched-chain α-ketoacids. OADCs were shown to be localized in close proximity to membrane-embedded respirasomes, as observed both in densely packed lamellar cristae of cardiac mitochondria and in ruptured mitochondrial samples where the dense packing is absent. This suggests the specificity of the OADC-respirasome interaction, which allows localized NADH/NAD+ exchange between OADCs and complex I of the respiratory chain. The importance of this local coupling is based on OADCs being the link between respiration, glycolysis and amino acid metabolism. The coupling of these basic metabolic processes can vary in different tissues and conditions and may be involved in the development of various pathologies. The present study shows that this important and previously missing parameter of mitochondrial complex coupling can be successfully assessed using cryo-electron tomography.

Integrated metabolomics and proteomics analysis to understand muscle atrophy resistance in hibernating Spermophilus dauricus.

Hibernating Spermophilus dauricus experiences minor muscle atrophy, which is an attractive anti-disuse muscle atrophy model. Integrated metabolomics and proteomics analysis was performed on the hibernating S. dauricus during the pre-hibernation (PRE) stage, torpor (TOR) stage, interbout arousal (IBA) stage, and post-hibernation (POST) stage. Time course stage transition-based (TOR vs. PRE, IBA vs. TOR, POST vs. IBA) differential expression analysis was performed based on the R limma package. A total of 14 co-differential metabolites were detected. Among these, l-cystathionine, l-proline, ketoleucine, serine, and 1-Hydroxy-3,6,7-Trimethoxy-2, 8-Diprenylxanthone demonstrated the highest levels in the TOR stage; Beta-Nicotinamide adenine dinucleotide, Dihydrozeatin, Pannaric acid, and Propionylcarnitine demonstrated the highest levels in the IBA stage; Adrenosterone, PS (18:0/14,15-EpETE), S-Carboxymethylcysteine, TxB2, and 3-Phenoxybenzylalcohol demonstrated the highest levels in the POST stage. Kyoto Encyclopedia of Genes and Genomes pathways annotation analysis indicated that biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism were co-differential metabolism pathways during the different stages of hibernation. The stage-specific metabolism processes and integrated enzyme-centered metabolism networks in the different stages were also deciphered. Overall, our findings suggest that (1) the periodic change of proline, ketoleucine, and serine contributes to the hindlimb lean tissue preservation; and (2) key metabolites related to the biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism may be associated with muscle atrophy resistance. In conclusion, our co-differential metabolites, co-differential metabolism pathways, stage-specific metabolism pathways, and integrated enzyme-centered metabolism networks are informative for biologists to generate hypotheses for functional analyses to perturb disuse-induced muscle atrophy.

Reshaping the Substrate Binding Pocket of ß-Amino Acid Dehydrogenase for the Synthesis of Aromatic ß-Amino Acids.

By reshaping the substrate-binding pocket of ß-amino acid dehydrogenase (ß-AADH), some variants were obtained with up to 2560-fold enhanced activity toward the model substrates (S)-ß-homophenylalanine and (R)-ß-phenylalanine. A few aromatic ß-amino acids were prepared with >99% ee and high isolated yields via either kinetic resolution of racemates or reductive amination of the corresponding ß-keto acids. This work expands the catalytic capability of ß-AADHs and highlights their practical application in the synthesis of pharmaceutically relevant ß-amino acids.

LC-MS/MS method for quantitative profiling of ketone bodies, α-keto acids, lactate, pyruvate and their stable isotopically labelled tracers in human plasma: An analytical panel for clinical metabolic kinetics and interactions.

An important area within clinical research is in vivo metabolism of ketone bodies (ß-hydroxybutyrate and acetoacetate) and in connection metabolites that may affect their production and/or cellular transport such as the keto-acids from the branched-chain amino acids, lactate and pyruvate. To determine in vivo metabolite turnover, availability of accurate and sensitive methods for analyzing the plasma concentrations of these metabolites and their stable isotopically labeled enrichments is mandatory. Therefore, the present study describes a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous analysis of ketone bodies, α-keto acids, lactate, pyruvate, and their tracer enrichments in humans using 2 different derivatization techniques with 4-bromo-N-methylbenzylamine and O-benzylhydroxylamine as derivatization reagents, and 1-ethyl-3-dimethylaminopropyl carbodiimide as coupling compound followed by a single LC-MS/MS run. The method was validated for matrix effects, linearity, accuracy, precision, recovery, stability, and enrichment (ratio) analysis of a stable isotopically labelled analytes (tracers) continuously infused in humans divided by the unlabeled endogenous analyte (tracee) that makes it possible to quantify the analyte in vivo synthesis and degradation rates. The applied parallel derivatization procedure yielded good sensitivity for all analytes of interest and their tracers. Despite the double derivatization method, mixing the ethyl acetate portions at the final stage made it possible to simultaneously analyze all compounds in a single LC-MS/MS run. Moreover, the liquid chromatography method was optimized to robustly quantify the keto acids derived from leucine (α-keto-isocaproic acid) and isoleucine (α-keto-ß-methylvaleric acid), the compounds with similar chemical structure and identical molecular weights. The presented method is designed and validated for human plasma. However, care should be taken in blood sampling and processing procedures as well as quick freezing and storage at -80 °C due to the instability of especially acetoacetate.

Recognizing and Managing a Metabolic Crisis.

In some relatively common inborn errors of metabolism there can be the accumulation of toxic compounds including ammonia and organic acids such as lactate and ketoacids, as well as energy deficits at the cellular level. The clinical presentation is often referred to as a metabolic emergency or crisis. Fasting and illness can result in encephalopathy within hours, and without appropriate recognition and intervention, the outcome may be permanent disability or death. This review outlines easy and readily available means of recognizing and diagnosing a metabolic emergency as well as general guidelines for management. Disease-specific interventions focus on parenteral nutrition to reverse catabolism, toxin removal strategies, and vitamin/nutrition supplementation.

A biocatalytic platform for asymmetric alkylation of α-keto acids by mining and engineering of methyltransferases.

Catalytic asymmetric α-alkylation of carbonyl compounds represents a long-standing challenge in synthetic organic chemistry. Herein, we advance a dual biocatalytic platform for the efficient asymmetric alkylation of α-keto acids. First, guided by our recently obtained crystal structures, we develop SgvMVAV as a general biocatalyst for the enantioselective methylation, ethylation, allylation and propargylation of a range of α-keto acids with total turnover numbers (TTNs) up to 4,600. Second, we mine a family of bacterial HMTs from Pseudomonas species sharing less than 50% sequence identities with known HMTs and evaluated their activities in SAM regeneration. Our best performing HMT from P. aeruginosa, PaHMT, displays the highest SAM regeneration efficiencies (TTN up to 7,700) among HMTs characterized to date. Together, the synergistic use of SgvMVAV and PaHMT affords a fully biocatalytic protocol for asymmetric methylation featuring a record turnover efficiency, providing a solution to the notorious problem of asymmetric alkylation.

Branched-chain ketoacid co-ingestion with protein lowers amino acid oxidation during hemodialysis: A randomized controlled cross-over trial.

BACKGROUND & AIMS: Hemodialysis removes amino acids from the circulation, thereby stimulating muscle proteolysis. Protein ingestion during hemodialysis can compensate for amino acid removal but may also increase uremic toxin production. Branched-chain ketoacid (BCKA) co-ingestion may provide an additional anabolic stimulus without adding to uremic toxin accumulation. In the present study we assessed the impact of BCKA co-ingestion with protein on forearm amino acid balance and amino acid oxidation during hemodialysis. METHODS: Nine patients (age: 73 ± 10 y) on chronic hemodialysis participated in this crossover trial. During two 4-h hemodialysis sessions, patients ingested 18 g protein with (PRO + BCKA) or without (PRO) 9 g BCKAs in a randomized order. Test beverages were labeled with L-[ring-13C6]-phenylalanine and provided throughout the last 3 h of hemodialysis as 18 equal sips consumed with 10-min intervals. Arterial and venous plasma as well as breath samples were collected frequently throughout hemodialysis. RESULTS: Arterial plasma total amino acid (TAA) concentrations during PRO and PRO + BCKA treatments were significantly lower after 1 h of hemodialysis (2.6 ± 0.3 and 2.6 ± 0.3 mmol/L, respectively) when compared to pre-hemodialysis concentrations (4.2 ± 1.0 and 4.0 ± 0.5 mmol/L, respectively; time effect: P < 0.001). Arterial plasma TAA concentrations increased throughout test beverage ingestion (time effect: P = 0.027) without differences between treatments (time∗treatment: P = 0.62). Forearm arteriovenous TAA balance during test beverage ingestion did not differ between timepoints (time effect: P = 0.31) or treatments (time∗treatment: P = 0.34). Whole-body phenylalanine oxidation was 33 ± 16% lower during PRO + BCKA when compared to PRO treatments (P < 0.001). CONCLUSIONS: BCKA co-ingestion with protein during hemodialysis does not improve forearm net protein balance but lowers amino acid oxidation.

Opine dehydrogenases, an underexplored enzyme family for the enzymatic synthesis of chiral amines.

Opines and opine-type chemicals are valuable natural products with diverse biochemical roles, and potential synthetic building blocks of bioactive compounds. Their synthesis involves reductive amination of ketoacids with amino acids. This transformation has high synthetic potential in producing enantiopure secondary amines. Nature has evolved opine dehydrogenases for this chemistry. To date, only one enzyme has been used as biocatalyst, however, analysis of the available sequence space suggests more enzymes to be exploited in synthetic organic chemistry. This review summarizes the current knowledge of this underexplored enzyme class, highlights key molecular, structural, and catalytic features with the aim to provide a comprehensive general description of opine dehydrogenases, thereby supporting future enzyme discovery and protein engineering studies.

Sensitive LC-MS/MS method for the temporal profiling of bile acids, fatty acids and branched-chain alpha-keto acids in maternal plasma during pregnancy and cord blood plasma at delivery.

BACKGROUND AND AIMS: There are significant changes to the maternal inflammatory profile across pregnancy. Recent studies suggest that perturbations in maternal gut microbial and dietary-derived plasma metabolites over the course of pregnancy mediate inflammation through a complex interplay of immunomodulatory effects. Despite this body of evidence, there is currently no analytical method that is suitable for the simultaneous profiling of these metabolites within human plasma. MATERIALS AND METHODS: We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the high-throughput analysis of these metabolites in human plasma without derivatization. Plasma samples were processed using liquid-liquid extraction method with varying proportions of methyl tert-butyl ether, methanol, and water in a 3:10:2.5 ratio to reduce matrix effects. RESULTS: LC-MS/MS detection was sufficiently sensitive to quantify these gut microbial and dietary-derived metabolites at physiological concentrations and linear calibration curves with r2 > 0.99 were obtained. Recovery was consistent across concentration levels. Stability experiments confirmed that up to 160 samples could be analyzed within a single batch. The method was validated and applied to analyse maternal plasma during the first and third trimester and cord blood plasma of 5 mothers. CONCLUSION: This study validated a straightforward and sensitive LC-MS/MS method for the simultaneous quantitation of gut microbial and dietary-derived metabolites in human plasma within 9 minutes without prior sample derivatization.