RESUMEN
l-Homocysteine, formed from S-adenosyl methionine following demethylation and adenosine release, accumulates when the methionine recycling pathway and other pathways become impaired, thus leading to hyperhomocysteinemia, a biomarker in cardiovascular diseases, neurological/psychiatric disorders, and cancer. The partial oxidation of the l-homocysteine thiol group and its decarboxylation on C-alpha lead to the formation of l-homocysteinesulfinic acid (l-HCSA) and homohypotaurine (HHT), respectively. Both compounds are not readily available from commercial suppliers, which hinders the investigation of their biological activities. Herein, the chemical synthesis of l-HCSA, from l-homocystine, was the starting point for establishing the bio-based synthesis of HHT using recombinant Escherichia coli glutamate decarboxylase (EcGadB), an enzyme already successfully employed for the bio-based synthesis of GABA and its phosphinic analog. Prior to HHT synthesis, kcat (33.92 ± 1.07) and KM (38.24 ± 3.45 mM) kinetic constants were determined for l-HCSA on EcGadB. The results of our study show that the EcGadB-mediated synthesis of HHT can be achieved with good yields (i.e., 40% following enzymatic synthesis and column chromatography). Purified HHT was tested in vitro on primary human umbilical vein endothelial cells and rat cardiomyoblasts and compared to the fully oxidized analog, homotaurine (OT, also known as tramiprosate), in widespread pharmaceutical use. The results show that both cell lines display statistically significant recovery from the cytotoxic effects induced by H2O2 in the presence of HHT.
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Escherichia coli , Glutamato Descarboxilasa , Homocisteína , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Humanos , Homocisteína/análogos & derivados , Homocisteína/metabolismo , Glutamato Descarboxilasa/metabolismo , Glutamato Descarboxilasa/genética , Proteínas Recombinantes/metabolismo , CinéticaRESUMEN
The bioavailability of natural folates is 50% lower than that of synthetic folic acid (FA); however, it remains unclear whether this value is universally applicable to all foods. Therefore, the present study investigated the bioavailability of folate from spinach using multiple biomarkers in a folate depletion-repletion mouse model. Mice were fed a folate-deficient diet for 4 wk and subsequently divided into three groups: folate-deficient, FA, and spinach folate. The folate repletion group received either FA or spinach folate at 2 mg/kg diet for 9 d. On the 7th day of repletion, half of each group underwent low-dose total body X-ray irradiation to induce chromosomal damage in bone marrow. Folate bioavailability biomarkers included measurements of folate levels in plasma, liver, and bone marrow along with an analysis of plasma homocysteine levels and chromosome damage, both of which are functional biomarkers of body folate. The consumption of a folate-deficient diet led to decreased tissue folate levels, increased plasma homocysteine levels, and chromosomal damage. Repletion with spinach folate restored folate levels in plasma, liver, and bone marrow to 69, 13, and 68%, respectively, of FA levels. Additionally, spinach folate repletion reduced plasma homocysteine levels and chromosome damage to 83% and 93-117%, respectively, of FA levels. Collectively, the present results demonstrated that the bioavailability of spinach folate exceeded 83% of FA, particularly when assessed using functional biomarkers.
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Disponibilidad Biológica , Biomarcadores , Deficiencia de Ácido Fólico , Ácido Fólico , Homocisteína , Hígado , Spinacia oleracea , Animales , Spinacia oleracea/química , Ácido Fólico/sangre , Biomarcadores/sangre , Deficiencia de Ácido Fólico/metabolismo , Hígado/metabolismo , Ratones , Masculino , Homocisteína/sangre , Homocisteína/metabolismo , Médula Ósea/metabolismo , Dieta , Modelos Animales de EnfermedadRESUMEN
Elevated levels of homocysteine (Hcy) and related metabolites are associated with Alzheimer's disease (AD). Severe hyperhomocysteinemia causes neurological deficits and worsens behavioral and biochemical traits associated with AD. Although Hcy is precluded from entering the Genetic Code by proofreading mechanisms of aminoacyl-tRNA synthetases, and thus is a non-protein amino acid, it can be attached to proteins via an N-homocysteinylation reaction mediated by Hcy-thiolactone. Because N-homocysteinylation is detrimental to a protein's function and biological integrity, Hcy-thiolactone-detoxifying enzymes-PON1, BLMH, BPHL-have evolved. This narrative review provides an account of the biological function of these enzymes and of the consequences of their impairments, leading to the phenotype characteristic of AD. Overall, accumulating evidence discussed in this review supports a hypothesis that Hcy-thiolactone contributes to neurodegeneration associated with a dysregulated Hcy metabolism.
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Enfermedad de Alzheimer , Homocisteína , Humanos , Enfermedad de Alzheimer/metabolismo , Homocisteína/metabolismo , Homocisteína/análogos & derivados , Animales , Hiperhomocisteinemia/metabolismoRESUMEN
Plasma nonesterified fatty acids (NEFA) are elevated in cancer, because of decreased albumin levels and of fatty acid oxidation, and increased fatty acid synthesis and lipolysis. Albumin depletion and NEFA elevation maximally release albumin-bound tryptophan (Trp) and increase its flux down the kynurenine pathway, leading to increased production of proinflammatory kynurenine metabolites, which tumors use to undermine T-cell function and achieve immune escape. Activation of the aryl hydrocarbon receptor by kynurenic acid promotes extrahepatic Trp degradation by indoleamine 2,3-dioxygenase and leads to upregulation of poly (ADP-ribose) polymerase, activation of which and also of SIRT1 (silent mating type information regulation 2 homolog 1) could lead to depletion of NAD+ and ATP, resulting in cell death. NEFA also modulate heme synthesis and degradation, changes in which impact homocysteine metabolism and production of reduced glutathione and hydrogen sulphide. The significance of the interactions between heme and homocysteine metabolism in cancer biology has received little attention. Targeting Trp disposition in cancer to prevent the NEFA effects is suggested.
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Ácidos Grasos no Esterificados , Neoplasias , Triptófano , Humanos , Neoplasias/metabolismo , Triptófano/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Animales , Receptores de Hidrocarburo de Aril/metabolismo , Quinurenina/metabolismo , Hemo/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Homocisteína/metabolismoRESUMEN
Biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play distinct yet crucial roles in various mitochondrial physiological activities. However, due to their similar chemical structures, distinguishing and detecting Cys/Hcy/GSH poses a considerable challenge. In this study, we developed a dual-channel, mitochondrial-targeted fluorescent probe termed QX-NBD, designed specifically for discriminating Cys/Hcy from GSH. The incorporation of a quinolinium group endowed the probe with excellent mitochondrial targeting capabilities. This functionality arose from the positively charged group's ability to selectively bind to negatively charged mitochondrial membranes through electrostatic interactions. Additionally, the ether bond between 4-chloro-7-nitro-1,2,3-benzoxadiazole and the near-infrared fluorophore QX-OH rendered the probe susceptible to nucleophilic attack by biothiols. Upon the introduction of Cys/Hcy, the probe exhibited dual fluorescence emissions in red and green. Conversely, the presence of GSH resulted in only red fluorescence emission. The detection limits of the probe for Cys and Hcy at 542 nm in buffer solution were determined to be 0.044 µM and 0.042 µM, respectively. Similarly, the detection limit for all these biothiols was 0.028 µM at 678 nm. Furthermore, the response times for Cys/Hcy/GSH were recorded as 4.0 min, 5.5 min, and 9.5 min, respectively. Moreover, the probe was employed to monitor fluctuations in biothiol levels during oxidative stress in both HeLa cells and zebrafish, demonstrating its applicability and utility in biological contexts.
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Colorantes Fluorescentes , Homocisteína , Mitocondrias , Pez Cebra , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Animales , Humanos , Mitocondrias/metabolismo , Mitocondrias/química , Células HeLa , Homocisteína/análisis , Homocisteína/metabolismo , Homocisteína/análogos & derivados , Compuestos de Sulfhidrilo/análisis , Compuestos de Sulfhidrilo/química , Glutatión/análisis , Glutatión/metabolismo , Cisteína/análisis , Espectrometría de Fluorescencia/métodos , Límite de Detección , Imagen Óptica/métodosRESUMEN
Elevated homocysteine (Hcy) levels are detrimental to neuronal cells and contribute to cognitive dysfunction in rats. Mitochondria plays a crucial role in cellular energy metabolism. Interestingly, the damaging effects of Hcy in vivo and in vitro conditions exhibit distinct results. Herein, we aimed to investigate the effects of Hcy on mitochondrial function in primary neurons and PC12 cells and explore the underlying mechanisms involved. The metabolic intermediates of Hcy act as methyl donors and play important epigenetic regulatory roles. N6-methyldeoxyadenosine (6 mA) modification, which is enriched in mitochondrial DNA (mtDNA), can be mediated by methylase METTL4. Our study suggested that mitochondrial perturbation caused by Hcy in primary neurons and PC12 cells may be attributable to mtDNA 6 mA modification difference. Hcy could activate the expression of METTL4 within mitochondria to facilitate mtDNA 6 mA status, and repress mtDNA transcription, then result in mitochondrial dysfunction.
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Desoxiadenosinas , Hipocampo , Homocisteína , Mitocondrias , Neuronas , Animales , Ratas , Células PC12 , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Homocisteína/farmacología , Homocisteína/análogos & derivados , Homocisteína/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Desoxiadenosinas/farmacología , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Ratas Sprague-Dawley , ADN Mitocondrial/metabolismo , ADN Mitocondrial/genética , Células Cultivadas , Metiltransferasas/metabolismo , Metiltransferasas/genéticaRESUMEN
Homocysteine thiolactone (HTL), a toxic metabolite of homocysteine (Hcy) in hyperhomocysteinemia (HHcy), is known to modify protein structure and function, leading to protein damage through formation of N-Hcy-protein. HTL has been highly linked to HHcy-associated cardiovascular and neurodegenerative diseases. The protective role of HTL hydrolases against HTL-associated vascular toxicity and neurotoxicity have been reported. Although several endogeneous enzymes capable of hydrolyzing HTL have been identified, the primary enzyme responsible for its metabolism remains unclear. In this study, three human carboxylesterases were screened to explore new HTL hydrolase and human carboxylesterase 1 (hCES1) demonstrates the highest catalytic activity against HTL. Given the abundance of hCES1 in the liver and the clinical significance of its single-nucleotide polymorphisms (SNPs), six common hCES1 nonsynonymous coding SNP (nsSNPs) variants were examined and characterized for their kinetic parameters. Variants E220G and G143E displayed 7.3-fold and 13.2-fold lower catalytic activities than its wild-type counterpart. In addition, the detailed catalytic mechanism of hCES1 for HTL hydrolysis was computational investigated and elucidated by Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) method. The function of residues E220 and G143 in sustaining its hydrolytic activity of hCES1 was analyzed, and the calculated energy difference aligns well with experimental-derived results, supporting the validity of our computational insights. These findings provide insights into the potential protective role of hCES1 against HTL-associated toxicity, and warrant future studies on the possible association between specific genetic variants of hCES1 with impaired catalytic function and clinical susceptibility of HTL-associated cardiovascular and neurodegenerative diseases.
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Homocisteína , Polimorfismo de Nucleótido Simple , Humanos , Homocisteína/metabolismo , Homocisteína/química , Homocisteína/análogos & derivados , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , CinéticaRESUMEN
Cystathionine beta-synthase-deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. HCU can be treated by using betaine to lower tissue and plasma levels of homocysteine (Hcy). Here, we show that mice with severely elevated Hcy and potentially deficient in the folate species tetrahydrofolate (THF) exhibit a very limited response to betaine indicating that THF plays a critical role in treatment efficacy. Analysis of a mouse model of HCU revealed a 10-fold increase in hepatic levels of 5-methyl -THF and a 30-fold accumulation of formiminoglutamic acid, consistent with a paucity of THF. Neither of these metabolite accumulations were reversed or ameliorated by betaine treatment. Hepatic expression of the THF-generating enzyme dihydrofolate reductase (DHFR) was significantly repressed in HCU mice and expression was not increased by betaine treatment but appears to be sensitive to cellular redox status. Expression of the DHFR reaction partner thymidylate synthase was also repressed and metabolomic analysis detected widespread alteration of hepatic histidine and glutamine metabolism. Many individuals with HCU exhibit endothelial dysfunction. DHFR plays a key role in nitric oxide (NO) generation due to its role in regenerating oxidized tetrahydrobiopterin, and we observed a significant decrease in plasma NOx (NO2 + NO3) levels in HCU mice. Additional impairment of NO generation may also come from the HCU-mediated induction of the 20-hydroxyeicosatetraenoic acid generating cytochrome CYP4A. Collectively, our data shows that HCU induces dysfunctional one-carbon metabolism with the potential to both impair betaine treatment and contribute to multiple aspects of pathogenesis in this disease.
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Homocistinuria , Hígado , Oxidación-Reducción , Tetrahidrofolato Deshidrogenasa , Tetrahidrofolatos , Animales , Homocistinuria/metabolismo , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Ratones , Tetrahidrofolatos/metabolismo , Hígado/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Betaína/metabolismo , Betaína/farmacología , Homocisteína/metabolismo , Ratones Endogámicos C57BL , Cistationina betasintasa/metabolismo , Cistationina betasintasa/genética , Carbono/metabolismo , Masculino , Ácido Fólico/metabolismo , FemeninoRESUMEN
Discovery and identification of a new endogenous metabolite are typically hindered by requirements of large sample volumes and multistage purifications to guide synthesis of the standard. Presented here is a metabolomics platform that uses chemical tagging and tandem mass spectrometry to determine structure, direct synthesis, and confirm identity. Three new homocysteine metabolites are reported: N-succinyl homocysteine, 2-methyl-1,3-thiazinane-4-carboxylic acid (MTCA), and homolanthinone.
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Homocisteína , Espectrometría de Masas en Tándem , Homocisteína/análisis , Homocisteína/metabolismo , Homocisteína/química , Espectrometría de Masas en Tándem/métodos , Metabolómica/métodos , Humanos , Tiazinas/químicaRESUMEN
Whether the long-term treatment of patients with proton pump inhibitors (PPIs) with different diseases [GERD, Zollinger-Ellison syndrome (ZES), etc.] can result in vitamin B12 (VB12) deficiency is controversial. In this study, in 175 patients undergoing long-term ZES treatment with anti-acid therapies, drug-induced control acid secretory rates were correlated with the presence/absence of VB12 deficiency, determined by assessing serum VB12 levels, measurements of VB12 body stores (blood methylmalonic acid (MMA) and total homocysteine[tHYC]), and other features of ZES. After a mean of 10.2 yrs. of any acid treatment (5.6 yrs. with PPIs), 21% had VB12 deficiency with significantly lower serum and body VB12 levels (p < 0.0001). The presence of VB12 deficiency did not correlate with any feature of ZES but was associated with a 12-fold lower acid control rate, a 2-fold higher acid control pH (6.4 vs. 3.7), and acid control secretory rates below those required for the activation of pepsin (pH > 3.5). Over a 5-yr period, the patients with VB12 deficiency had a higher rate of achlorhydria (73% vs. 24%) and a lower rate of normal acid secretion (0% vs. 49%). In conclusion, in ZES patients, chronic long-term PPI treatment results in marked acid hyposecretion, resulting in decreased serum VB12 levels and decreased VB12-body stores, which can result in VB12 deficiency.
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Inhibidores de la Bomba de Protones , Deficiencia de Vitamina B 12 , Vitamina B 12 , Síndrome de Zollinger-Ellison , Humanos , Inhibidores de la Bomba de Protones/efectos adversos , Inhibidores de la Bomba de Protones/uso terapéutico , Deficiencia de Vitamina B 12/tratamiento farmacológico , Síndrome de Zollinger-Ellison/tratamiento farmacológico , Femenino , Masculino , Persona de Mediana Edad , Adulto , Vitamina B 12/sangre , Anciano , Ácido Metilmalónico/sangre , Homocisteína/sangre , Homocisteína/metabolismoRESUMEN
Elevation of the homocysteine concentration in the plasma called hyperhomocysteinemia (hHCY) during pregnancy causes a number of pre- and postnatal developmental disorders. The aim of our study was to analyze the effects of H2S donors -NaHS and N-acetylcysteine (NAC) on blood-brain barrier (BBB) permeability in rats with prenatal hHCY. In rats with mild hHCY BBB permeability assessed by Evans Blue extravasation in brain increased markedly throughout life. Administration of NaHS or NAC during pregnancy attenuated hHCY-associated damage and increased endogenous concentrations of sulfides in brain tissues. Acute application of dl-homocysteine thiolactone induced BBB leakage, which was prevented by the NMDA receptor antagonist MK-801 or H2S donors. Rats with hHCY demonstrated high levels of NO metabolite - nitrites and proinflammatory cytokines (IL-1ß, TNF-α, IL-6) in brain. Lactate dehydrogenase (LDH) activity in the serum was higher in rats with hHCY. Mitochondrial complex-I activity was lower in brain of hHCY rats. NaHS treatment during pregnancy restored levels of proinflammatory cytokines, nitrites and activity of the respiratory chain complex in brain as well as the LDH activity in serum. Our data suggest that H2S has neuroprotective effects against prenatal hHCY-associated BBB disturbance providing a potential strategy for the prevention of developmental impairments in newborns.
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Acetilcisteína , Barrera Hematoencefálica , Citocinas , Sulfuro de Hidrógeno , Hiperhomocisteinemia , Fármacos Neuroprotectores , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Embarazo , Hiperhomocisteinemia/metabolismo , Femenino , Sulfuro de Hidrógeno/metabolismo , Fármacos Neuroprotectores/farmacología , Acetilcisteína/farmacología , Citocinas/metabolismo , Homocisteína/sangre , Homocisteína/metabolismo , Homocisteína/análogos & derivados , Ratas Wistar , Sulfuros/farmacología , Sulfuros/administración & dosificación , Ratas , Masculino , Complicaciones del Embarazo , Encéfalo/metabolismo , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/sangre , Permeabilidad , Nitritos/metabolismo , Nitritos/sangreRESUMEN
BACKGROUND: Obstructive sleep apnea (OSA) is associated with hypertension. However, the differential mechanisms underlying OSA-related hypertension between normal-weight vs. obese patients is limited. METHODS: We studied 92 patients with OSA and 24 patients with continuous positive airway pressure (CPAP) treatment. Blood pressure (BP) was measured twice during awake and continuously monitored during sleep. Obesity was defined as body mass index ≥28 kg/m2. Serum metabolite levels were assessed by metabolomics. RESULTS: Among 59 normal-weight and 33 obese patients, 651 and 167 metabolites showed differences between hypertension and normotension or were associated with systolic and diastolic BP (SBP, DBP) after controlling confounders. These metabolites involved 16 and 12 Kyoto Encyclopedia of Genes and Genomes enrichment pathways in normal-weight and obese patients respectively, whereas 6 pathways overlapped. Among these 6 overlapping pathways, 4 were related to homocysteine metabolism and 2 were non-specific pathways. In homocysteine metabolism pathway, 13 metabolites were identified. Interestingly, the change trends of 7 metabolites associated with SBP (all interaction-p≤0.083) and 8 metabolites associated with DBP (all interaction-p≤0.033) were opposite between normal-weight and obese patients. Specifically, increased BP was associated with down-regulated folate-dependent remethylation and accelerated transsulfuration in normal-weight patients, whereas associated with enhanced betaine-dependent remethylation and reduced transsulfuration in obese patients. Similar findings were observed in ambulatory BP during sleep. After CPAP treatment, baseline low homocysteine levels predicted greater decrease in DBP among normal-weight but not obese patients. CONCLUSIONS: Mechanisms in OSA-related hypertension differ between normal-weight and obese patients, which are explained by different changes in homocysteine metabolism.
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Presión de las Vías Aéreas Positiva Contínua , Homocisteína , Hipertensión , Obesidad , Apnea Obstructiva del Sueño , Humanos , Apnea Obstructiva del Sueño/terapia , Apnea Obstructiva del Sueño/complicaciones , Apnea Obstructiva del Sueño/fisiopatología , Apnea Obstructiva del Sueño/metabolismo , Homocisteína/sangre , Homocisteína/metabolismo , Masculino , Obesidad/complicaciones , Obesidad/metabolismo , Femenino , Persona de Mediana Edad , Adulto , Presión Sanguínea/fisiología , Índice de Masa CorporalRESUMEN
Folate is a water-soluble B vitamin involved in the synthesis of purines and pyrimidines and is one of the essential vitamins for human growth and reproduction. Folate deficiency due to low dietary intake, poor absorption of folate, and alterations in folate metabolism due to genetic defects or drug interactions significantly increases the risk of diseases such as neural tube defects, cardiovascular disease, cancer, and cognitive dysfunction. Recent studies have shown that folate deficiency can cause hyperhomocysteinemia, which increases the risk of hypertension and cardiovascular disease, and that high homocysteine levels are an independent risk factor for liver fibrosis and cirrhosis. In addition, folate deficiency results in increased secretion of pro-inflammatory factors and impaired lipid metabolism in the liver, leading to lipid accumulation in hepatocytes and fibrosis. There is substantial evidence that folate deficiency contributes to the development and progression of a variety of liver diseases, including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), viral hepatitis, hepatic fibrosis, and liver cancer. Here we review key studies on the role of folate in the pathophysiology of liver diseases, summarize the current status of studies on folate in the treatment of liver diseases, and speculate that folate may be a potential therapeutic target for liver diseases.
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Deficiencia de Ácido Fólico , Ácido Fólico , Hepatopatías , Enfermedad del Hígado Graso no Alcohólico , Humanos , Ácido Fólico/metabolismo , Hepatopatías/metabolismo , Hepatopatías/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Animales , Neoplasias Hepáticas/metabolismo , Hiperhomocisteinemia , Homocisteína/metabolismo , Metabolismo de los LípidosRESUMEN
PURPOSE: There is currently no ideal radiotracer for imaging bacterial infections. Radiolabelled D-amino acids are promising candidates because they are actively incorporated into the peptidoglycan of the bacterial cell wall, a structural feature which is absent in human cells. This work describes fluorine-18 labelled analogues of D-tyrosine and D-methionine, O-(2-[18F]fluoroethyl)-D-tyrosine (D-[18F]FET) and S-(3-[18F]fluoropropyl)-D-homocysteine (D-[18F]FPHCys), and their pilot evaluation studies as potential radiotracers for imaging bacterial infection. PROCEDURES: D-[18F]FET and D-[18F]FPHCys were prepared in classical fluorination-deprotection reactions, and their uptake in Staphylococcus aureus and Pseudomonas aeruginosa was evaluated over 2 h. Heat killed bacteria were used as controls. A clinically-relevant foreign body model of S. aureus infection was established in Balb/c mice, as well as a sterile foreign body to mimic inflammation. The ex vivo biodistribution of D-[18F]FPHCys in the infected and inflamed mice was evaluated after 1 h, by dissection and gamma counting. The uptake was compared to that of [18F]FDG. RESULTS: In vitro uptake of both D-[18F]FET and D-[18F]FPHCys was specific to live bacteria. Uptake was higher in S. aureus than in P. aeruginosa for both radiotracers, and of the two, higher for D-[18F]FPHCys than D-[18F]FET. Blocking experiments with non-radioactive D-[19F]FPHCys confirmed specificity of uptake. In vivo, D-[18F]FPHCys had greater accumulation in S. aureus infection compared with sterile inflammation, which was statistically significant. As anticipated, [18F]FDG showed no significant difference in uptake between infection and inflammation. CONCLUSIONS: D-[18F]FPHCys uptake was higher in infected tissues than inflammation, and represents a fluorine-18 labelled D-AA with potential to detect a S. aureus reference strain (Xen29) in vivo. Additional studies are needed to evaluate uptake of this radiotracer in clinical isolates.
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Ratones Endogámicos BALB C , Tomografía de Emisión de Positrones , Staphylococcus aureus , Tirosina , Animales , Tomografía de Emisión de Positrones/métodos , Proyectos Piloto , Tirosina/análogos & derivados , Tirosina/química , Distribución Tisular , Homocisteína/metabolismo , Homocisteína/análogos & derivados , Homocisteína/química , Pseudomonas aeruginosa , Radiofármacos/química , Radiofármacos/farmacocinética , Radioisótopos de Flúor/química , Femenino , Ratones , Infecciones Bacterianas/diagnóstico por imagen , Infecciones Bacterianas/microbiologíaRESUMEN
Chinese hamster ovary (CHO) cells require cysteine for growth and productivity in fed-batch cultures. In intensified processes, supplementation of cysteine at high concentrations is a challenge due to its limited solubility and instability in solution. Methionine can be converted to cysteine (CYS) but key enzymes, cystathionine beta-synthase (Cbs) and cystathionine gamma-lyase (Cth), are not active in CHO cells resulting in accumulation of an intermediate, homocysteine (HCY), in cell culture milieu. In this study, Cbs and Cth were overexpressed in CHO cells to confer cysteine prototrophy, i.e., the ability to grow in a cysteine free environment. These pools (CbCt) needed homocysteine and beta-mercaptoethanol (ßME) to grow in CYS-free medium. To increase intracellular homocysteine levels, Gnmt was overexpressed in CbCt pools. The resultant cell pools (GnCbCt), post adaptation in CYS-free medium with decreasing residual HCY and ßME levels, were able to proliferate in the HCY-free, ßME-free and CYS-free environment. Interestingly, CbCt pools were also able to be adapted to grow in HCY-free and CYS-free conditions, albeit at significantly higher doubling times than GnCbCt cells, but couldn't completely adapt to ßME-free conditions. Further, single cell clones derived from the GnCbCt cell pool had a wide range in expression levels of Cbs, Cth and Gnmt and, when cultivated in CYS-free fed-batch conditions, performed similarly to the wild type (WT) cell line cultivated in CYS supplemented fed-batch culture. Intracellular metabolomic analysis showed that HCY and glutathione (GSH) levels were lower in the CbCt pool in CYS-free conditions but were restored closer to WT levels in the GnCbCt cells cultivated in CYS-free conditions. Transcriptomic analysis showed that GnCbCt cells upregulated several genes encoding transporters as well as methionine catabolism and transsulfuration pathway enzymes that support these cells to biosynthesize cysteine effectively. Further, 'omics analysis suggested CbCt pool was under ferroptotic stress in CYS-free conditions, which, when inhibited, enhanced the growth and viability of these cells in CYS-free conditions.
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Cricetulus , Cisteína , Ingeniería Metabólica , Células CHO , Animales , Cisteína/metabolismo , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Cricetinae , Homocisteína/metabolismo , Homocisteína/genéticaRESUMEN
Elevated homocysteine (Hcy) levels have been recognized as significant risk factor for cardiovascular and cerebrovascular diseases, closely related to endothelial injury. While expression of Ciliary Neurotrophic Factor (CNTF) significantly increases during Hcy-induced vascular endothelial cell injury, the precise molecular pathways through which CNTF operates remain to be clarified. To induce vascular endothelial cell injury, human umbilical vein endothelial cells (HUVECs) were treated with Hcy. Cell viability and apoptosis in HUVECs were assessed using the CCK-8 assay and flow cytometry. Western blot analysis determined the expression levels of the JAK2-STAT3 pathway, inflammation-related factors (IL-1ß, NLRP3, ICAM-1, VCAM-1), and apoptosis-related factors (cleaved Caspase-3 and Bax). Immunofluorescence staining and western blotting were employed to examine CD31 and α-SMA expression. Knockdown of CNTF was achieved using lentiviral interference, and its effects on inflammation and cell injury were evaluated. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter analysis were conducted to investigate the interaction between the MAFK and CNTF promoters. Our results indicated that Hcy induced high expression of CNTF and activated the JAK2-STAT3 signaling pathway, thereby upregulating factors associated with inflammation and cell apoptosis. Inhibiting CNTF alleviated Hcy-induced inflammation and cell injury. MAFK was identified as a transcription factor promoting CNTF transcription, and its overexpression exacerbated inflammation and cell injury in Hcy-treated HUVECs through the CNTF-JAK2-STAT3 axis, which could be reversed by knocking down CNTF. Activation of MAFK leads to CNTF upregulation, which activates the JAK2-STAT3 signaling pathway, regulating inflammation and inducing injury in Hcy-exposed vascular endothelial cells. Targeting CNTF or its upstream regulator MAFK may represent potential therapeutic strategies for mitigating endothelial dysfunction associated with hyperhomocysteinemia and cardiovascular diseases.
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Apoptosis , Factor Neurotrófico Ciliar , Homocisteína , Células Endoteliales de la Vena Umbilical Humana , Inflamación , Janus Quinasa 2 , Factor de Transcripción STAT3 , Transducción de Señal , Janus Quinasa 2/metabolismo , Humanos , Factor de Transcripción STAT3/metabolismo , Homocisteína/farmacología , Homocisteína/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Inflamación/metabolismo , Inflamación/patología , Factor Neurotrófico Ciliar/metabolismo , Factor Neurotrófico Ciliar/genética , Apoptosis/efectos de los fármacos , Células Cultivadas , Supervivencia Celular/efectos de los fármacosRESUMEN
AIMS: This study aimed to evaluate the association between gestational diabetes mellitus (GDM) and circulating folate metabolites, folic acid (FA) intake, and the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genotype. MATERIALS AND METHODS: A prospective pregnancy cohort study was conducted in Beijing, China, from 2022 to 2023. Circulating folate metabolites, including red blood cell (RBC) 5-methyltetrahydrofolate (5-MTHF), 5, 10-methylene-tetrahydrofolate (5,10-CH2-THF), 5- formyltetrahydrofolate (5-CHO-THF), and unmetabolised folic acid (UMFA), and plasma homocysteine (HCY), 5-MTHF, and methylmalonic acid (MMA), were determined at 6-17 weeks and 20-26 weeks of gestation. FA intake and the MTHFR and MTRR genotype were also examined. GDM was diagnosed between 24 and 28 weeks of pregnancy by a 75-g oral glucose tolerance test (OGTT). The association between the folate status and GDM was ascertained using multivariate generalised linear models, logistic regression models, and restricted cubic spline regression, adjusting for potential confounders. RESULTS: The study included 2032 pregnant women, of whom 392 (19.29%) developed GDM. UMFA above the 75th percentile (≥P75) [adjusted OR (aOR) (95% confidence interval [CI]) = 1.36 (1.01-1.84)], UMFA ≥ P90 [aOR (95% CI) = 1.82 (1.23-2.69)], and HCY ≥ P75 [aOR (95% CI) = 1.40 (1.04-1.88)] in early pregnancy, and RBC 5-MTHF [aOR (95% CI) = 1.48 (1.10-2.00)], RBC 5,10-CH2-THF [aOR (95% CI) = 1.55 (1.15-2.10)], and plasma 5-MTHF [aOR (95% CI) = 1.36 (1.00-1.86)] in mid-pregnancy ≥ P75 are associated with GDM. Higher UMFA levels in early pregnancy show positive associations with the 1-h and 2-h glucose levels during the OGTT, and higher HCY levels are associated with increased fasting glucose levels during the OGTT. In comparison, RBC 5- MTHF and 5,10-CH2-THF, and plasma 5- MTHF in mid-pregnancy are positively associated with the 1-h glucose level (p < 0.05). The MTHFR and MTRR genotype and FA intake are not associated with GDM. CONCLUSIONS: Elevated levels of UMFA and HCY during early pregnancy, along with elevated RBC 5-MTHF and 5,10-CH2-THF and plasma 5-MTHF during mid-pregnancy, are associated with GDM. These findings indicate distinct connections between different folate metabolites and the occurrence of GDM.
Asunto(s)
Diabetes Gestacional , Ácido Fólico , Metilenotetrahidrofolato Reductasa (NADPH2) , Humanos , Femenino , Diabetes Gestacional/sangre , Diabetes Gestacional/metabolismo , Embarazo , Ácido Fólico/sangre , Estudios Prospectivos , Adulto , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Biomarcadores/sangre , Estudios de Seguimiento , Ferredoxina-NADP Reductasa/genética , Genotipo , China/epidemiología , Pronóstico , Segundo Trimestre del Embarazo/sangre , Homocisteína/sangre , Homocisteína/metabolismoRESUMEN
INTRODUCTION: Although previous studies have linked obesity and erectile dysfunction, the novel surrogate indicators of adipose accumulation are more essential and dependable factors to consider. Therefore, the primary objective of the current investigation was to examine and clarify the association between metabolic score for visceral fat (METS-VF) and erectile dysfunction. METHODS: Firstly, multivariate logistic regression analysis, smoothed curve fitting, and threshold effect analysis were employed to investigate the association between METS-VF and erectile dysfunction. Mediation analysis was also performed to evaluate the mediating role of homocysteine and inflammation. After that, subgroup analysis was carried out to examine the stability of the correlation of METS-VF with erectile dysfunction in various population settings. Furthermore, the area under the receiver operating characteristic (ROC) curve and eXtreme Gradient Boosting (XGBoost) algorithm were utilized to assess the capability of identifying METS-VF in comparison to the other four obesity-related indicators in identifying erectile dysfunction. RESULTS: After adjusting for all confounding factors, METS-VF was strongly and favourablely correlated with erectile dysfunction. With each additional unit rise in METS-VF, the prevalence of erectile dysfunction increased by 141%. A J-shaped relationship between METS-VF and erectile dysfunction was discovered through smoothed curve fitting. Marital status, physical activity, and smoking status can potentially modify this association. This finding of the ROC curve suggests that METS-VF had a powerful identifying capacity for erectile dysfunction (AUC = 0.7351). Homocysteine and inflammation mediated 4.24% and 2.81%, respectively. CONCLUSION: The findings of the current investigation suggest that METS-VF can be considered a dependable identifying indicator of erectile dysfunction.
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Disfunción Eréctil , Curva ROC , Masculino , Disfunción Eréctil/metabolismo , Disfunción Eréctil/fisiopatología , Humanos , Persona de Mediana Edad , Grasa Intraabdominal/metabolismo , Grasa Intraabdominal/patología , Biomarcadores/metabolismo , Adulto , Homocisteína/sangre , Homocisteína/metabolismo , Obesidad/complicaciones , Obesidad/metabolismo , Anciano , Factores de Riesgo , Síndrome Metabólico/complicaciones , Síndrome Metabólico/metabolismo , Modelos LogísticosRESUMEN
One-carbon (1-C) metabolic deficiency impairs homeostasis, driving disease development, including infertility. It is of importance to summarize the current evidence regarding the clinical utility of 1-C metabolism-related biomolecules and methyl donors, namely, folate, betaine, choline, vitamin B12, homocysteine (Hcy), and zinc, as potential biomarkers, dietary supplements, and culture media supplements in the context of medically assisted reproduction (MAR). A narrative review of the literature was conducted in the PubMed/Medline database. Diet, ageing, and the endocrine milieu of individuals affect both 1-C metabolism and fertility status. In vitro fertilization (IVF) techniques, and culture conditions in particular, have a direct impact on 1-C metabolic activity in gametes and embryos. Critical analysis indicated that zinc supplementation in cryopreservation media may be a promising approach to reducing oxidative damage, while female serum homocysteine levels may be employed as a possible biomarker for predicting IVF outcomes. Nonetheless, the level of evidence is low, and future studies are needed to verify these data. One-carbon metabolism-related processes, including redox defense and epigenetic regulation, may be compromised in IVF-derived embryos. The study of 1-C metabolism may lead the way towards improving MAR efficiency and safety and ensuring the lifelong health of MAR infants.
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Carbono , Técnicas Reproductivas Asistidas , Humanos , Carbono/metabolismo , Vitamina B 12/metabolismo , Fertilización In Vitro/métodos , Femenino , Homocisteína/metabolismo , Homocisteína/sangre , Ácido Fólico/metabolismo , Suplementos Dietéticos , Colina/metabolismo , Zinc/metabolismo , Betaína/metabolismo , BiomarcadoresRESUMEN
Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.