Enhancement of Exfoliating Effects through the Novel Cosmetic Ingredient Mandelic acid_Carnitine Ion-Pairing Complex.

PURPOSE: This study aimed to develop a novel exfoliating material with high efficacy and low irritation by synthesizing the Mandelic acid_Carnitine ion pairing complex (M_C complex) and evaluating its exfoliating properties. Additionally, the study assessed the skin improvement effects of the M_C complex through clinical evaluations. METHODS: The M_C complex was synthesized in a 1:1 molar ratio of Mandelic acid and Carnitine. Structural characterization was performed using dynamic light scattering and Fourier-transform infrared spectroscopy. Exfoliating efficacy was evaluated on porcine skin, and clinical assessments were conducted on human subjects to measure various skin improvement parameters. RESULTS: The formation of the M_C complex was confirmed through particle size analysis, zeta-potential measurements, and FT-IR spectroscopy. The M_C complex demonstrated superior exfoliating efficacy compared to Mandelic acid alone, especially at pH 4.5. Clinical evaluations showed significant improvements in blackheads, whiteheads, pore volume, depth, density, count, and affected area, as well as skin texture. No adverse reactions were observed. CONCLUSION: The M_C complex exhibits high exfoliating efficacy and minimal irritation, making it a promising cosmetic ingredient for improving skin health. These findings support its potential as a low-irritation exfoliating material under mildly acidic conditions, contributing to overall skin health enhancement.

Biocompatible Tough Ionogels with Reversible Supramolecular Adhesion.

Cohesive and interfacial adhesion energies are difficult to balance to obtain reversible adhesives with both high mechanical strength and high adhesion strength, although various methods have been extensively investigated. Here, a biocompatible citric acid/L-(-)-carnitine (CAC)-based ionic liquid was developed as a solvent to prepare tough and high adhesion strength ionogels for reversible engineered and biological adhesives. The prepared ionogels exhibited good mechanical properties, including tensile strength (14.4 MPa), Young's modulus (48.1 MPa), toughness (115.2 MJ m-3), and high adhesion strength on the glass substrate (24.4 MPa). Furthermore, the ionogels can form mechanically matched tough adhesion at the interface of wet biological tissues (interfacial toughness about 191 J m-2) and can be detached by saline solution on demand, thus extending potential applications in various clinical scenarios such as wound adhesion and nondestructive transfer of organs.

An untargeted analytical workflow based on Kendrick mass defect filtering reveals dysregulations in acylcarnitines in prostate cancer tissue.

BACKGROUND: Metabolomics is nowadays considered one the most powerful analytical for the discovery of metabolic dysregulations associated with the insurgence of cancer, given the reprogramming of the cell metabolism to meet the bioenergetic and biosynthetic demands of the malignant cell. Notwithstanding, several challenges still exist regarding quality control, method standardization, data processing, and compound identification. Therefore, there is a need for effective and straightforward approaches for the untargeted analysis of structurally related classes of compounds, such as acylcarnitines, that have been widely investigated in prostate cancer research for their role in energy metabolism and transport and ß-oxidation of fatty acids. RESULTS: In the present study, an innovative analytical platform was developed for the straightforward albeit comprehensive characterization of acylcarnitines based on high-resolution mass spectrometry, Kendrick mass defect filtering, and confirmation by prediction of their retention time in reversed-phase chromatography. In particular, a customized data processing workflow was set up on Compound Discoverer software to enable the Kendrick mass defect filtering, which allowed filtering out more than 90 % of the initial features resulting from the processing of 25 tumoral and adjacent non-malignant prostate tissues collected from patients undergoing radical prostatectomy. Later, a partial least square-discriminant analysis model validated by repeated double cross-validation was built on the dataset of 74 annotated acylcarnitines, with classification rates higher than 93 % for both groups, and univariate statistical analysis helped elucidate the individual role of the annotated metabolites. SIGNIFICANCE: Hydroxylation of short- and medium-chain minor acylcarnitines appeared to be a significant variable in describing tissue differences, suggesting the hypothesis that the neoplastic growth is linked to oxidation phenomena on selected metabolites and reinforcing the need for effective methods for the annotation of minor metabolites.

New small-molecule alcohol synthesis by breaking the space limitation of the "aromatic cage" in Pseudomonas sp. AK1 BBOX.

Fe(II)/2OG-dependent oxygenase γ-butyrobetaine hydroxylase (BBOX) stereoselectively hydroxylates inactive C-H bonds and produces L-carnitine. It has potential applications in the biosynthesis of L-carnitine and the synthesis of other small molecule alcohols. In this paper, we systematically explore the substrate range of Pseudomonas sp. AK1 BBOX (psBBOX), with emphasis on the quaternary ammonium portion of γ-butyrobetaine (γ-BB). The space limitation of the "aromatic cage" in psBBOX in the hydroxylation of large quaternary ammonium analogues was studied, and the role of four aromatic amino acid residues in the substrate binding mode was analyzed. Consequently, the F188A mutant was developed with the ability to hydroxylate cyclic quaternary ammonium analogues and generate new alcohol compounds by breaking the limitation of the "aromatic cage".

Acylcarnitines: Nomenclature, Biomarkers, Therapeutic Potential, Drug Targets, and Clinical Trials.

Acylcarnitines are fatty acid metabolites that play important roles in many cellular energy metabolism pathways. They have historically been used as important diagnostic markers for inborn errors of fatty acid oxidation and are being intensively studied as markers of energy metabolism, deficits in mitochondrial and peroxisomal ß -oxidation activity, insulin resistance, and physical activity. Acylcarnitines are increasingly being identified as important indicators in metabolic studies of many diseases, including metabolic disorders, cardiovascular diseases, diabetes, depression, neurologic disorders, and certain cancers. The US Food and Drug Administration-approved drug L-carnitine, along with short-chain acylcarnitines (acetylcarnitine and propionylcarnitine), is now widely used as a dietary supplement. In light of their growing importance, we have undertaken an extensive review of acylcarnitines and provided a detailed description of their identity, nomenclature, classification, biochemistry, pathophysiology, supplementary use, potential drug targets, and clinical trials. We also summarize these updates in the Human Metabolome Database, which now includes information on the structures, chemical formulae, chemical/spectral properties, descriptions, and pathways for 1240 acylcarnitines. This work lays a solid foundation for identifying, characterizing, and understanding acylcarnitines in human biosamples. We also discuss the emerging opportunities for using acylcarnitines as biomarkers and as dietary interventions or supplements for many wide-ranging indications. The opportunity to identify new drug targets involved in controlling acylcarnitine levels is also discussed. SIGNIFICANCE STATEMENT: This review provides a comprehensive overview of acylcarnitines, including their nomenclature, structure and biochemistry, and use as disease biomarkers and pharmaceutical agents. We present updated information contained in the Human Metabolome Database website as well as substantial mapping of the known biochemical pathways associated with acylcarnitines, thereby providing a strong foundation for further clarification of their physiological roles.

Medium & long-chain acylcarnitine's relation to lipid metabolism as potential predictors for diabetic cardiomyopathy: a metabolomic study.

BACKGROUND: Acylcarnitine is an intermediate product of fatty acid oxidation. It is reported to be closely associated with the occurrence of diabetic cardiomyopathy (DCM). However, the mechanism of acylcarnitine affecting myocardial disorders is yet to be explored. This current research explores the different chain lengths of acylcarnitines as biomarkers for the early diagnosis of DCM and the mechanism of acylcarnitines for the development of DCM in-vitro. METHODS: In a retrospective non-interventional study, 50 simple type 2 diabetes mellitus patients and 50 DCM patients were recruited. Plasma samples from both groups were analyzed by high throughput metabolomics and cluster heat map using mass spectrometry. Principal component analysis was used to compare the changes occurring in the studied 25 acylcarnitines. Multivariable binary logistic regression was used to analyze the odds ratio of each group for factors and the 95% confidence interval in DCM. Myristoylcarnitine (C14) exogenous intervention was given to H9c2 cells to verify the expression of lipid metabolism-related protein, inflammation-related protein expression, apoptosis-related protein expression, and cardiomyocyte hypertrophy and fibrosis-related protein expression. RESULTS: Factor 1 (C14, lauroylcarnitine, tetradecanoyldiacylcarnitine, 3-hydroxyl-tetradecanoylcarnitine, arachidic carnitine, octadecanoylcarnitine, 3-hydroxypalmitoleylcarnitine) and factor 4 (octanoylcarnitine, hexanoylcarnitine, decanoylcarnitine) were positively correlated with the risk of DCM. Exogenous C14 supplementation to cardiomyocytes led to increased lipid deposition in cardiomyocytes along with the obstacles in adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathways and affecting fatty acid oxidation. This further caused myocardial lipotoxicity, ultimately leading to cardiomyocyte hypertrophy, fibrotic remodeling, and increased apoptosis. However, this effect was mitigated by the AMPK agonist acadesine. CONCLUSIONS: The increased plasma levels in medium and long-chain acylcarnitine extracted from factors 1 and 4 are closely related to the risk of DCM, indicating that these factors can be an important tool for DCM risk assessment. C14 supplementation associated lipid accumulation by inhibiting the AMPK/ACC/CPT1 signaling pathway, aggravated myocardial lipotoxicity, increased apoptosis apart from cardiomyocyte hypertrophy and fibrosis were alleviated by the acadesine.

Design of High-Relaxivity Polyelectrolyte Nanocapsules Based on Citrate Complexes of Gadolinium(III) of Unusual Composition.

Through nuclear magnetic relaxation and pH-metry, the details of the complexation of gadolinium(III) ions with citric acid (H4L) in water and aqueous solutions of cationic polyelectrolytes are established. It is shown that the presence of poly(ethylene imine) (PEI) in solution affects magnetic relaxation behavior of gadolinium(III) complexes with citric acid (Cit) to a greater extent than polydiallyldimethylammonium chloride (PDDC). A large increase in relaxivity (up to 50 mM-1s-1) in the broad pH range (4-8) is revealed for the gadolinium(III)-citric acid-PEI system, which is particularly strong in the case of PEI with the molecular weight of 25 and 60 kDa. In weakly acidic medium (pH 3-7), the presence of PEI results in the formation of two tris-ligand associates [Gd(H2L)3]3- and [Gd(H2L)2(HL)]4-, which do not exist in aqueous medium. In weakly alkaline medium (pH 7-10), formation of ternary complexes Gd(III)-Cit-PEI with the Gd(III)-to-Cit ratio of 1:2 is evidenced. Using transmission electron microscopy (TEM) and dynamic light scattering techniques (DLS), the formation of the particles with the size of 50-100 nm possessing narrow molecular-mass distribution (PDI 0.08) is determined in the solution containing associate of PEI with tris-ligand complex [Gd(H2L)2(HL)]4-.

The MttB superfamily member MtyB from the human gut symbiont Eubacterium limosum is a cobalamin-dependent γ-butyrobetaine methyltransferase.

The production of trimethylamine (TMA) from quaternary amines such as l-carnitine or γ-butyrobetaine (4-(trimethylammonio)butanoate) by gut microbial enzymes has been linked to heart disease. This has led to interest in enzymes of the gut microbiome that might ameliorate net TMA production, such as members of the MttB superfamily of proteins, which can demethylate TMA (e.g., MttB) or l-carnitine (e.g., MtcB). Here, we show that the human gut acetogen Eubacterium limosum demethylates γ-butyrobetaine and produces MtyB, a previously uncharacterized MttB superfamily member catalyzing the demethylation of γ-butyrobetaine. Proteomic analyses of E. limosum grown on either γ-butyrobetaine or dl-lactate were employed to identify candidate proteins underlying catabolic demethylation of the growth substrate. Three proteins were significantly elevated in abundance in γ-butyrobetaine-grown cells: MtyB, MtqC (a corrinoid-binding protein), and MtqA (a corrinoid:tetrahydrofolate methyltransferase). Together, these proteins act as a γ-butyrobetaine:tetrahydrofolate methyltransferase system, forming a key intermediate of acetogenesis. Recombinant MtyB acts as a γ-butyrobetaine:MtqC methyltransferase but cannot methylate free cobalamin cofactor. MtyB is very similar to MtcB, the carnitine methyltransferase, but neither was detectable in cells grown on carnitine nor was detectable in cells grown with γ-butyrobetaine. Both quaternary amines are substrates for either enzyme, but kinetic analysis revealed that, in comparison to MtcB, MtyB has a lower apparent Km for γ-butyrobetaine and higher apparent Vmax, providing a rationale for MtyB abundance in γ-butyrobetaine-grown cells. As TMA is readily produced from γ-butyrobetaine, organisms with MtyB-like proteins may provide a means to lower levels of TMA and proatherogenic TMA-N-oxide via precursor competition.

Carnitine is a pharmacological allosteric chaperone of the human lysosomal α-glucosidase.

Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient's enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.

Phosphate stress triggers the conversion of glycerol into l-carnitine in Pseudomonas fluorescens.

Glycerol, a by-product of the biofuel industry is transformed into l-carnitine when the soil microbe Pseudomonas fluorescens is cultured in a phosphate-limited mineral medium (LP). Although the biomass yield was similar to that recorded in phosphate-sufficient cultures (HP), the rate of growth was slower. Phosphate was completely consumed in the LP cultures while in the HP media, approximately 35 % of the initial phosphate was detected at stationary phase of growth. The enhanced production of α-ketoglutarate (KG) in HP cultures supplemented with manganese was recently reported (Alhasawi et al., 2017). l-carnitine appeared to be a prominent metabolite in the spent fluid while the soluble cellular-free extract was characterized with peaks attributable to lysine, γ-butyrobetaine (GB), acetate and succinate in the LP cultures. Upon incubation with glycerol and NH4Cl, the resting cells readily secreted l-carnitine and revealed the presence of such precursors like GB, lysine and methionine involved in the synthesis of this trimethylated moiety. Functional proteomic studies of select enzymes participating in tricarboxylic acid cycle (TCA), oxidative phosphorylation (OP), glyoxylate cycle and l-carnitine synthesis revealed a major metabolic reconfiguration evoked by phosphate stress. While isocitrate dehydrogenase-NAD+ dependent (ICDH-NAD+) and Complex I were markedly diminished, the activities of γ-butyrobetaine aldehyde dehydrogenase (GBADH) and l-carnitine dehydrogenase (CDH) were enhanced. Real-time quantitative polymerase chain reaction (RT-qPCR) analyses pointed to an increase in transcripts of the enzymes γ-butyrobetaine dioxygenase (bbox1), S-adenosylmethionine synthase (metK) and l-carnitine dehydrogenase (lcdH). The l-carnitine/γ-butyrobetaine antiporter (caiT) was enhanced more than 400-fold in the LP cultures compared to the HP controls. This metabolic reprogramming modulated by phosphate deprivation may provide an effective technology to transform glycerol, an industrial waste into valuable l-carnitine.

Prospective diagnosis of MT-ATP6-related mitochondrial disease by newborn screening.

Elevated citrulline and C5-OH levels are reported as part of the newborn screening of core and secondary disorders on the Recommended Uniform Screening Panel (RUSP). Additionally, some state laboratory newborn screening programs report low citrulline levels, which may be observed in proximal urea cycle disorders. We report six patients who were found on newborn screening to have low citrulline and/or elevated C5-OH levels in whom confirmatory testing showed the combination of these two abnormal analytes. Mitochondrial sequencing revealed known pathogenic variants in MT-ATP6 at high heteroplasmy levels in all cases. MT-ATP6 at these heteroplasmy levels is associated with Leigh syndrome, a progressive neurodegenerative disease. Patients were treated with supplemental citrulline and, in some cases, mitochondrial cofactor therapy. These six patients have not experienced metabolic crises or developmental regression, and early diagnosis and management may help prevent the neurological sequelae of Leigh syndrome. The affected mothers and siblings are asymptomatic or paucisymptomatic (e.g. intellectual disability, depression, migraines, obsessive-compulsive disorder, and poor balance) despite high heteroplasmy or apparent homoplasmy of the familial variant, thus expanding the clinical spectrum seen in pathogenic variants of MT-ATP6. Confirmatory plasma amino acid analysis and acylcarnitine profiling should be ordered in a patient with either low citrulline and/or elevated C5-OH, as this combination appears specific for pathogenic variants in MT-ATP6.

Quantitation and evaluation of perinatal medium-chain and long-chain acylcarnitine blood concentrations in 12,000 full-term breastfed newborns.

OBJECTIVES: Medium-chain (MCA) and long-chain acylcarnitine (LCA) blood concentrations play a significant role in the fatty acid (FA) oxidation process, especially during the first days of life. Identification of their abnormal concentrations, via expanded newborn screening, can lead to the diagnosis of FA oxidation disorders. This study aimed to demonstrate MCA and LCA concentrations in Dried Blood Spots (DBS) of full-term breastfed infants, in relation to their birth weight (BW) perinatally. METHODS: Breastfed full-term infants (n = 12,000, 6,000 males, 6,000 females) with BW 2,000-3,999 g were divided into four equal groups: Group A, 2,000-2,499 g, B 2,500-2,999 g, C 3,000-3,499 g, and D 3,500-3,999 g. Samples were collected as DBS and acylcarnitines were determined via a liquid chromatography tandem mass spectrometry method. RESULTS: MCA and LCA blood concentrations were determined significantly lower in group A (low birth weight infants) in both sexes. Infants with BW > 3,500 g (group D), were characterized by lower levels of C10, C10:1, C14, C14:1 acylcarnitines and higher levels of C16 and C18:1 acylcarnitines, as compared to the other groups of this study. CONCLUSIONS: Concentration patterns in full-term breastfed newborns in relation to sex and mainly BW found in this study could be very helpful for neonatologists, especially for newborns of group A.

Functionalities of chitosan conjugated with lauric acid and l-carnitine and application of the modified chitosan in an oil-in-water emulsion.

The aim of this research was to conjugate chitosan (CT) with lauric acid (LA) and l-carnitine (CNT) to yield a product that is water-soluble at neutral pH and has surface, antimicrobial, and antioxidant activities. The resulting CT-LA-CNT is water-soluble at neutral pH, in contrast with CT and CT-LA, which require the aid of acid to become soluble. Concerning antimicrobial activity, for S. aureus, the minimum bactericidal concentration of CT was lower than those of CT-LA or CT-LA-CNT, while the three compounds exhibited similar bactericidal activity against E. coli. CT-LA-CNT was also used to study the oxidative stability of soybean oil in an oil-in-water (O/W) emulsion; sodium dodecyl sulfate (SDS) and Tween 80 and Span 80 (TS), an emulsifier mixture, were used as controls for comparison. The results showed that CT-LA-CNT was better than SDS and TS at protecting the lipid from oxidation.

The Mitochondrial Carnitine Acyl-carnitine Carrier (SLC25A20): Molecular Mechanisms of Transport, Role in Redox Sensing and Interaction with Drugs.

The SLC25A20 transporter, also known as carnitine acyl-carnitine carrier (CAC), catalyzes the transport of short, medium and long carbon chain acyl-carnitines across the mitochondrial inner membrane in exchange for carnitine. The 30-year story of the protein responsible for this function started with its purification from rat liver mitochondria. Even though its 3D structure is not yet available, CAC is one of the most deeply characterized transport proteins of the inner mitochondrial membrane. Other than functional, kinetic and mechanistic data, post-translational modifications regulating the transport activity of CAC have been revealed. CAC interactions with drugs or xenobiotics relevant to human health and toxicology and the response of the carrier function to dietary compounds have been discovered. Exploiting combined approaches of site-directed mutagenesis with chemical targeting and bioinformatics, a large set of data on structure/function relationships have been obtained, giving novel information on the molecular mechanism of the transport catalyzed by this protein.

Graphene oxide and Lambda exonuclease assisted screening of L-carnitine aptamers and the site-directed mutagenesis design of C-rich structure aptamer.

In this study, Graphene Oxide (GO) was used to screen the binding with the aptamers of L-carnitine chiral enantiomers. The ssDNA library was prepared by the method of Lambda exonuclease. In addition, a simple casing device was designed to improve the purification and recovery efficiency of the small ssDNA fragments in the process of screening. Finally, more than 160,000 aptamer sequences were obtained by high-throughput sequencing. We determined the strongest affinity aptamer sequence, CA04, by the Resonance Rayleigh scattering (RRS) technology. We also analyzed the key binding sites (in the 16th position case) of the truncated aptamer sequence CAD10. Interestingly, we found that aptamer CA10 and CA06 were both C-rich bases through sequence alignment and analysis, and the aptamer CA10 was confirmed that the CA10 and CA06 were formed under acidic conditions (pH 4.5) by CD spectrum and ESI-MS analysis. The interaction between gold nanoparticles (AuNPs) and functionalized aptamer CA10 was analyzed. We used Site-directed mutagenesis design and QGRS Mapper to optimize aptamer CA10, where an optimal aptamer CA10-03 were obtained after affinity analysis. It is also proved to be an effective method to obtain stronger affinity aptamer. Meanwhile, Native-PAGE and UV spectrum analysis were performed on the mutation sequences, and the interaction with ThT was analyzed. Finally, it is hoped that my study can provide help for later identification and detection of L-carnitine.

Neuroprotective Effects of Carnitine and Its Potential Application to Ameliorate Neurotoxicity.

Carnitine is an essential metabolite that is absorbed from the diet and synthesized in the kidney, liver, and brain. It ferries fatty acids across the mitochondrial membrane to undergo ß-oxidation. Carnitine has been studied as a therapy or protective agent for many neurological diseases and neurotoxicity (e.g., prolonged anesthetic exposure-induced developmental neurotoxicity in preclinical models). Preclinical and clinical data support the notion that carnitine or acetyl carnitine may improve a patient's quality of life through increased mitochondrial respiration, release of neurotransmitters, and global gene expression changes, showing the potential of carnitine beyond its approved use to treat primary and secondary carnitine deficiency. In this review, we summarize the beneficial effects of carnitine or acetyl carnitine on the central nervous system, highlighting protective effects against neurotoxicity-induced damage caused by various chemicals and encouraging a thorough evaluation of carnitine use as a therapy for patients suffering from neurotoxicant exposure.

Safety indicators of a novel multi supplement based on guarana, selenium, and L-carnitine: Evidence from human and red earthworm immune cells.

Neurodegenerative diseases are associated with chronic inflammatory states. There is evidence to support the design of novel supplements based on guarana (G) (Paullinia cupana), selenium (S), and L-carnitine (C), the use of which, potentially attenuates neuro oxi-inflammatory conditions. Therefore, this study analyzed the cytotoxic and redox effects of GSC on human leucocytes, the inflammatory activation of microglia BV-2 cells, and effect on mortality, oxidative metabolism, and the immune modulation of red earthworms (Eisenia fetida). The GSC concentrations tested in cell culture were in the range of 0.04-2.1 mg/mL. All the GSC-supplemented samples tested, reverted H2O2 oxidation in DNA molecules, suggesting its genoprotective potential. GSC did not induce mortality in leucocyte cultures. On the contrary, a reduction in the levels of oxidation of lipids, proteins, and cell apoptosis was observed, via downregulation of caspase 3 and 8 genes. GSC showed a dual effect on microglia, decreasing the cellular proliferation at lower concentrations (<0.24 mg/mL) and increasing the cellular proliferation mainly at concentrations > 1.0 mg/mL. GSC did not have a toxic effect on red earthworms, but induced an increase in amoebocyte cells and in brown body formation, indicating immune response activation. The results suggest that GSC could be safe for human consumption.

Light-Activated Electron Transfer and Catalytic Mechanism of Carnitine Oxidation by Rieske-Type Oxygenase from Human Microbiota.

Oxidation of quaternary ammonium substrate, carnitine by non-heme iron containing Acinetobacter baumannii (Ab) oxygenase CntA/reductase CntB is implicated in the onset of human cardiovascular disease. Herein, we develop a blue-light (365 nm) activation of NADH coupled to electron paramagnetic resonance (EPR) measurements to study electron transfer from the excited state of NADH to the oxidized, Rieske-type, [2Fe-2S]2+ cluster in the AbCntA oxygenase domain with and without the substrate, carnitine. Further electron transfer from one-electron reduced, Rieske-type [2Fe-2S]1+ center in AbCntA-WT to the mono-nuclear, non-heme iron center through the bridging glutamate E205 and subsequent catalysis occurs only in the presence of carnitine. The electron transfer process in the AbCntA-E205A mutant is severely affected, which likely accounts for the significant loss of catalytic activity in the AbCntA-E205A mutant. The NADH photo-activation coupled with EPR is broadly applicable to trap reactive intermediates at low temperature and creates a new method to characterize elusive intermediates in multiple redox-centre containing proteins.

Enhancing confidence of metabolite annotation in Capillary Electrophoresis-Mass Spectrometry untargeted metabolomics with relative migration time and in-source fragmentation.

Capillary electrophoresis coupled to mass spectrometry is a power tool in untargeted metabolomics studies to analyze charged and polar compounds. However, identification is a challenge due to the variability of migration times and the lack of MS/MS spectra in CE-TOF-MS, the type of instruments most frequently employed. We present here a CE-MS search platform incorporated in CEU Mass Mediator to annotate metabolites with a confidence level L2. For its the development we analyzed 226 compounds using two fragmentor voltages: 100 and 200 V. The information obtained, such as relative migration times (RMT) and in-source fragments, were incorporated into the platform. In addition, we validated the CE-MS search functionality using different types of biological samples such as plasma samples (human, rat, and rabbit), mouse macrophages, and human urine. The RMT tolerance percentage for the search of metabolites has been determined, establishing 5% for all compounds, except for the compounds migrating in the electro-osmotic flow, for which the tolerance should be of 10%. It has also been demonstrated the robustness of the in-source fragmentation, which makes possible the annotation of compounds by means of their fragmentation pattern. As an example, 3-methylhistidine and 1-methilhistidine, whose RMT are very close, have been annotated. Studies of the fragmentation mechanisms of acyl-L-carnitines have shown that in-source fragmentation follows the general fragmentation rules and is a suitable alternative to MS/MS.

Walnut Consumption, Plasma Metabolomics, and Risk of Type 2 Diabetes and Cardiovascular Disease.

BACKGROUND: Walnut consumption is associated with lower risk of type 2 diabetes (T2D) and cardiovascular disease (CVD). However, it is unknown whether plasma metabolites related to walnut consumption are also associated with lower risk of cardiometabolic diseases. OBJECTIVES: The study aimed to identify plasma metabolites associated with walnut consumption and evaluate the prospective associations between the identified profile and risk of T2D and CVD. METHODS: The discovery population included 1833 participants at high cardiovascular risk from the PREvención con DIeta MEDiterránea (PREDIMED) study with available metabolomics data at baseline. The study population included 57% women (baseline mean BMI (in kg/m2): 29.9; mean age: 67 y). A total of 1522 participants also had available metabolomics data at year 1 and were used as the internal validation population. Plasma metabolomics analyses were performed using LC-MS. Cross-sectional associations between 385 known metabolites and walnut consumption were assessed using elastic net continuous regression analysis. A 10-cross-validation (CV) procedure was used, and Pearson correlation coefficients were assessed between metabolite weighted models and self-reported walnut consumption in each pair of training-validation data sets within the discovery population. We further estimated the prospective associations between the identified metabolite profile and incident T2D and CVD using multivariable Cox regression models. RESULTS: A total of 19 metabolites were significantly associated with walnut consumption, including lipids, purines, acylcarnitines, and amino acids. Ten-CV Pearson correlation coefficients between self-reported walnut consumption and the plasma metabolite profile were 0.16 (95% CI: 0.11, 0.20) in the discovery population and 0.15 (95% CI: 0.10, 0.20) in the validation population. The metabolite profile was inversely associated with T2D incidence (HR per 1 SD: 0.83; 95% CI: 0.71, 0.97; P = 0.02). For CVD incidence, the HR per 1-SD was 0.71 (95% CI: 0.60, 0.85; P < 0.001). CONCLUSIONS: A metabolite profile including 19 metabolites was associated with walnut consumption and with a lower risk of incident T2D and CVD in a Mediterranean population at high cardiovascular risk.