RESUMO
In this paper, the development and application of a multiple heart-cutting achiral-chiral LC-LC method (mLC-LC) for the analysis of dansylated (Dns) branched-chain amino acids in commercial tablets are described. In the first dimension, a Waters Xbridge RP C18 achiral column was used under gradient conditions with buffered aqueous solution and acetonitrile. The elution order Dns-valine (Dns-Val) < Dns-isoleucine (Dns-Ile) < Dns-leucine (Dns-Leu) turned out with full resolution between adjacent peaks: 7.25 and 1.50 for the Val/Ile and the Ile/Leu pairs, respectively. A "research" validation study was performed, revealing high accuracy (Recovery%) and precision (RSD%) using two external set solutions, respectively, in the range 93.7%-104.1% and 0.4%-3.2%. The C18 column was connected via a two-position six-port switching valve to the quinidine-based Chiralpak quinidine-anion-exchange chiral column. A water/acetonitrile, 30/70 (v/v) with 50 mM ammonium acetate (apparent pH of 5.5) eluent allowed getting the three enantiomers' pairs resolved: RS equal to 4.3 for Dns-Val and Dns-Ile, and 1.7 for Dns-Leu. The application of the mLC-LC method confirmed that the content of Val, Ile, and Leu in the tablets was compliant with that labeled by the producer. Only l-enantiomers were found in the food supplement, as confirmed by LC-MS/MS analysis.
Assuntos
Aminoácidos de Cadeia Ramificada , Comprimidos , Comprimidos/química , Aminoácidos de Cadeia Ramificada/análise , Aminoácidos de Cadeia Ramificada/química , Estereoisomerismo , Cromatografia Líquida/métodos , Reprodutibilidade dos Testes , Compostos de Dansil/química , Espectrometria de Massas em Tandem/métodos , Modelos LinearesRESUMO
Elevations in circling branched-chain amino acids (BCAAs) levels associated with insulin resistance and type 2 diabetes mellitus (T2DM). Morus alba L. water extracts (MLE) show hypoglycemic function, but the precise mechanism remains obscure. This study is designed to investigate the association of the antidiabetes effect of MLE with the BCAAs co-metabolism modulated by host and gut microbiota. Tissue-specific expressions of BCAA-catabolizing enzymes were detected by RT-PCR and western blot, respectively. The components of the intestinal microflora were analyzed by high-throughput 16S rRNA gene sequencing. The results showed that MLE administration improved blood glucose and insulin level, decreased inflammatory cytokines expression, and lowered serum and feces BCAAs levels. Furthermore, MLE reversed the abundance changes of the bacterial genera correlated with serum and feces BCAAs, such as Anaerovorax, Bilophila, Blautia, Colidextribacter, Dubosiella, Intestinimonas, Lachnoclostridium, Lachnospiraceae_NK4A136, Oscillibacter, and Roseburia. Functionality prediction indicated that MLE potentially inhibited bacterial BCAAs biosynthesis, and promoted the tissue-specific expression of BCAAs catabolic enzyme. More importantly, MLE had obvious impacts on BCAA catabolism in germ-free-mimic T2DM mice. Those results indicated that MLE improving T2DM-related biochemical abnormalities is associated with not only gut microbiota modification but also the tissue-specific expression of BCAAs catabolic enzyme.
Assuntos
Diabetes Mellitus Tipo 2 , Microbioma Gastrointestinal , Resistência à Insulina , Morus , Camundongos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Morus/química , RNA Ribossômico 16S/análise , Aminoácidos de Cadeia Ramificada/análise , Aminoácidos de Cadeia Ramificada/metabolismo , Folhas de Planta/químicaRESUMO
Chinese hamster ovary (CHO) cells in fed-batch cultures produce several metabolic byproducts derived from amino acid catabolism, some of which accumulate to growth inhibitory levels. Controlling the accumulation of these byproducts has been shown to significantly enhance cell proliferation. Interestingly, some of these byproducts have physiological roles that go beyond inhibition of cell proliferation. In this study, we show that, in CHO cell fed-batch cultures, branched-chain amino acid (BCAA) catabolism contributes to the formation of butyrate, a novel byproduct that is also a well-established specific productivity enhancer. We further show that other byproducts of BCAA catabolism, namely isovalerate and isobutyrate, which accumulate in CHO cell fed-batch cultures, also enhance specific productivity. Lastly, we show that the rate of production of these BCAA catabolic byproducts is negatively correlated with glucose uptake and lactate production rates. Thus, limiting glucose supply to suppress glucose uptake and lactate production, as in the case of fed-batch cultures employing high-end pH-controlled delivery of glucose (HiPDOG) technology, significantly enhances BCAA catabolic byproduct accumulation, resulting in higher specific productivities.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Butiratos/metabolismo , Aminoácidos de Cadeia Ramificada/análise , Animais , Reatores Biológicos , Butiratos/análise , Células CHO , Cricetinae , Cricetulus , Meios de Cultura/química , Meios de Cultura/metabolismo , Glucose/metabolismoRESUMO
In the presented study, a capillary electrophoresis-mass spectrometry method combining high separation efficiency and sensitive detection has been developed and validated, for the first time, to quantify branched chain amino acids (valine, isoleucine, leucine) in commercial food and sport supplement samples and human plasma samples. The separations were performed in a bare fused silica capillary. The background electrolyte was composed of 500 mM formic acid with pH 2.0. The plasma sample pretreatment was realized by simple protein precipitation with acetonitrile. Injection of a short zone of highly basic electrolyte before the sample injection and application of the negative pressure on the separation were accompanied by enhanced resolution of the isobaric amino acids-isoleucine and leucine. The developed method was characterized by favorable validation parameters, such as linearity (r2 > 0.99), accuracy and precision, the limit of detection, lower limit of quantification, or robustness. These parameters were more than sufficient for the quantification of branched chain amino acids in various samples. The determined concentrations of branched chain amino acids in food and sports supplements were in very good agreement with the content declared by the manufacturer. The investigated concentrations of branched chain amino acids were in the range 294.68-359.24 µM for valine, 91.76-95.67 µM for isoleucine, and 196.78-251.24 µM for leucine. These concentrations fall within the physiological limits. The developed CE-MS/MS method represents a suitable alternative to traditional approaches used in branched chain amino acid quality control and bioanalysis.
Assuntos
Aminoácidos de Cadeia Ramificada/análise , Sangue/metabolismo , Suplementos Nutricionais , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Adulto , Aminoácidos de Cadeia Ramificada/sangue , Análise Química do Sangue/métodos , Humanos , MasculinoRESUMO
Due to the utilization of milk proteins such as whey protein (WP) and casein as sports nutrition ergogenic aids, the present study investigated the effects of the association of WP and casein in a ratio of 80:20, a similar ratio of human breast milk, on blood branched-chain amino acid (BCAA) profiles, markers of protein metabolism and delayed onset muscle soreness (DOMS), after a single bout of resistance exercise. A double-blind, crossover and acute study was carried out with ten men (age 29 ± 8 years; BMI: 25.4 ± 2.9 kg/m2; 77 ± 12 kg; 1.74 ± 0.09 m); each one consumed the following supplements randomly, one per session: WP, CAS (casein), WP/CAS (80% WP/20% CAS), CAS/WP (80% CAS/20% WP) and PLA (placebo). They were also subjected to the following evaluations: the one repetition maximum (1RM) test; resistance training session; blood extraction during each session to determine the BCAA profile; two food records; 3-day evaluation of DOMS (24 h, 48 h and 72 h) and nitrogen balance in each treatment. The intervention resulted in similar nitrogen urinary, creatinine and urea plasma levels and showed a positive nitrogen balance in all the trials. Regarding the BCAAs, the peak occurred at 60 min post-ingestion and remained higher until 120 min for WP, WP/CAS and CAS/WP. The DOMS was significantly lower for WP, WP/CAS and CAS/WP compared to the CAS and PLA treatments. There were no advantages in the association of WP and CAS in the BCAAs profile when compared to WP itself, but it induced a lower DOMS compared to CAS and PLA (Clinical Trial registration number: clinicaltrials.gov, NCT04648384).
Assuntos
Caseínas/análise , Exercício Físico/fisiologia , Leite Humano/química , Proteínas do Soro do Leite/análise , Adulto , Aminoácidos de Cadeia Ramificada/análise , Biomarcadores/metabolismo , Humanos , Masculino , Mialgia/patologiaRESUMO
INTRODUCTION: Studies have shown that systemic levels of branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) are elevated in cardiometabolic diseases (CMDs) in populations resident in high income countries. However, little is known about the association of BCAAs and AAAs with metabolic syndrome and its components in Asian Indian (AI) and Black African (BA) populations. OBJECTIVE: The aim of this study was to describe the association of BCAAs and AAAs with the metabolic syndrome, its individual components and insulin resistance in AI and BA populations. METHODS: Serum samples collected from AI (n = 349) and BA (n = 369) subjects were used to measure levels of BCAAs and AAAs by ultra-pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Anthropometric, demographic and cardiometabolic variables were measured in all subjects. RESULTS: The sum of BCAAs and AAAs was higher in AIs compared to BAs. The BCAAs and AAAs were positively associated with insulin resistance, metabolic syndrome and its individual components. This was particularly the case for AI subjects, in unadjusted regression models. However, these associations were non-significant after adjusting for co-variates, particularly visceral adipose tissue (VAT). Triglyceride levels were significantly associated with valine and leucine levels in BAs even after adjustment for co-variates. Lastly, we found that fasting circulatory BCAA and AAA levels are strongly correlated with VAT in both populations. CONCLUSION: This study identified specific associations of serum valine and leucine levels with triglycerides in BAs. The association of amino acids with CMDs was observed in AIs, but was found to be the result of confounding by VAT. Further studies are required to determine whether BCAAs and AAAs are aetiological factors in CMDs and how VAT modulates their serum levels.
Assuntos
Aminoácidos Aromáticos/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Doenças Cardiovasculares/metabolismo , Adulto , África/epidemiologia , Aminoácidos Aromáticos/análise , Aminoácidos de Cadeia Ramificada/análise , Povo Asiático , População Negra , Glicemia/metabolismo , Fatores de Risco Cardiometabólico , Doenças Cardiovasculares/prevenção & controle , Feminino , Humanos , Masculino , Síndrome Metabólica/metabolismo , Síndrome Metabólica/fisiopatologia , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Feeding pigs with very-low protein (VLP) diets while supplemented with limiting amino acids (AA) results in decreased growth. The objective of this study was to determine if supplementing VLP diets with branched-chain AA (BCAA) would reverse the negative effects of these diets on growth and whether this is associated with alterations in energy balance, blood metabolomics and fecal microbiota composition. Twenty-four nursery pigs were weight-matched, individually housed and allotted into following treatments (n = 8/group): control (CON), low protein (LP) and LP supplemented with BCAA (LP + BCAA) for 4 weeks. Relative to CON, pigs fed with LP had lower feed intake (FI) and body weight (BW) throughout the study, but those fed with LP + BCAA improved overall FI computed for 4 weeks, tended to increase the overall average daily gain, delayed the FI and BW depression for ~ 2 weeks and had transiently higher energy expenditure. Feeding pigs with LP + BCAA impacted the phenylalanine and protein metabolism and fatty acids synthesis pathways. Compared to CON, the LP + BCAA group had higher abundance of Paludibacteraceae and Synergistaceae and reduced populations of Streptococcaceae, Oxyphotobacteria_unclassified, Pseudomonadaceae and Shewanellaceae in their feces. Thus, supplementing VLP diets with BCAA temporarily annuls the adverse effects of these diets on growth, which is linked with alterations in energy balance and metabolic and gut microbiome profile.
Assuntos
Aminoácidos de Cadeia Ramificada/análise , Dieta com Restrição de Proteínas , Suplementos Nutricionais/análise , Metabolismo Energético/efeitos dos fármacos , Fezes/microbiologia , Metabolômica , Microbiota/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Citocinas/sangue , Ingestão de Alimentos/efeitos dos fármacos , Respiração/efeitos dos fármacos , SuínosRESUMO
Oncogenic drivers of progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) such as c-MYC have downstream effects on intracellular metabolic pathways of clonal plasma cells (PCs). Thus, extracellular environments such as the bone marrow (BM) plasma likely have unique metabolite profiles that differ from patients with MGUS compared to MM. This study utilized an untargeted metabolite and targeted complex lipid profiling of BM plasma to identify significant differences in the relative metabolite levels between patients with MGUS and MM from an exploratory cohort. This was followed by verification of some of the metabolite differences of interest by targeted quantification of the metabolites using isotopic internal standards in the exploratory cohort as well as an independent validation cohort. Significant differences were noted in the amino acid profiles such as decreased branch chain amino acids (BCAAs) and increased catabolism of tryptophan to the active kynurenine metabolite 3-hydroxy-kynurenine between patients with MGUS and MM. A decrease in the total levels of complex lipids such as phosphatidylethanolamines (PE), lactosylceramides (LCER) and phosphatidylinositols (PI) were also detected in the BM plasma samples from MM compared to MGUS patients. Thus, metabolite and complex lipid profiling of the BM plasma identifies differences in levels of metabolites and lipids between patients with MGUS and MM. This may provide insight into the possible differences of the intracellular metabolic pathways of their clonal PCs.
Assuntos
Metabolômica/métodos , Gamopatia Monoclonal de Significância Indeterminada/diagnóstico , Mieloma Múltiplo/diagnóstico , Plasmócitos/metabolismo , Aminoácidos de Cadeia Ramificada/análise , Diagnóstico Diferencial , Humanos , Cinurenina/análise , Lactosilceramidas/análise , Lipidômica/métodos , Gamopatia Monoclonal de Significância Indeterminada/metabolismo , Mieloma Múltiplo/sangue , Mieloma Múltiplo/metabolismo , Fosfatidiletanolaminas/análise , Fosfatidilinositóis/análise , Estudos ProspectivosRESUMO
Mucopolysaccharidoses (MPSs) are inherited disorders of the glycosaminoglycan (GAG) metabolism. The defective digestion of GAGs within the intralysosomal compartment of affected patients leads to a broad spectrum of clinical manifestations ranging from cardiovascular disease to neurological impairment. The molecular mechanisms underlying the progression of the disease downstream of the genetic mutation of genes encoding for lysosomal enzymes still remain unclear. Here, we applied a targeted metabolomic approach to a mouse model of PS IIIB, using a platform dedicated to the diagnosis of inherited metabolic disorders, in order to identify amino acid and fatty acid metabolic pathway alterations or the manifestations of other metabolic phenotypes. Our analysis highlighted an increase in the levels of branched-chain amino acids (BCAAs: Val, Ile, and Leu), aromatic amino acids (Tyr and Phe), free carnitine, and acylcarnitines in the liver and heart tissues of MPS IIIB mice as compared to the wild type (WT). Moreover, Ala, Met, Glu, Gly, Arg, Orn, and Cit amino acids were also found upregulated in the liver of MPS IIIB mice. These findings show a specific impairment of the BCAA and fatty acid catabolism in the heart of MPS IIIB mice. In the liver of affected mice, the glucose-alanine cycle and urea cycle resulted in being altered alongside a deregulation of the BCAA metabolism. Thus, our data demonstrate that an accumulation of BCAAs occurs secondary to lysosomal GAG storage, in both the liver and the heart of MPS IIIB mice. Since BCAAs regulate the biogenesis of lysosomes and autophagy mechanisms through mTOR signaling, impacting on lipid metabolism, this condition might contribute to the progression of the MPS IIIB disease.
Assuntos
Fígado/química , Metabolômica/métodos , Mucopolissacaridose III/metabolismo , Miocárdio/química , Aminoácidos Aromáticos/análise , Aminoácidos de Cadeia Ramificada/análise , Animais , Carnitina/análogos & derivados , Carnitina/análise , Modelos Animais de Doenças , Humanos , Metabolismo dos Lipídeos , Masculino , CamundongosRESUMO
Silverskin, the main coffee roasting by-product, is rich in fiber, protein and antioxidants. Its protein fraction was studied regarding total, protein and non-protein nitrogen content. Amino acids were analysed after automated on-line derivatization. The method showed to be precise (<6.9%) and accurate (recoveries using a certified reference material and spiked blanks: 90-102%; for spiked samples: 73-113%). The real protein content of silverskin was 12%. One quarter of the total nitrogen corresponded to the non-protein fraction. All essential amino acids were present in the free form, except methionine. Regarding total amino acids, aspartic and glutamic acids (9-10 mg/g) were the major compounds. Branched chain amino acids (leucine, isoleucine and valine) were also present in substantial amounts (5-8 mg/g), as well as proline and arginine (~5 mg/g). Concluding, silverskin is a source of amino acids with relevance for the improvement of cognitive and physical performances.
Assuntos
Aminoácidos/análise , Coffea/química , Nitrogênio/análise , Proteínas de Plantas/análise , Resíduos/análise , Aminoácidos de Cadeia Ramificada/análise , Antioxidantes/análise , Extratos Vegetais/análise , SementesRESUMO
The tumorigenic role and underlying mechanisms of lipid accumulation, commonly observed in many cancers, remain insufficiently understood. In this study, we identified an AMP-activated protein kinase (AMPK)-GATA-binding protein 3 (GATA3)-enoyl-CoA hydratase short-chain 1 (ECHS1) pathway that induces lipid accumulation and promotes cell proliferation in clear cell renal cell carcinoma (ccRCC). Decreased expression of ECHS1, which is responsible for inactivation of fatty acid (FA) oxidation and activation of de novo FA synthesis, positively associated with ccRCC progression and predicted poor patient survival. Mechanistically, ECHS1 downregulation induced FA and branched-chain amino acid (BCAA) accumulation, which inhibited AMPK-promoted expression of GATA3, a transcriptional activator of ECHS1. BCAA accumulation induced activation of mTORC1 and de novo FA synthesis, and promoted cell proliferation. Furthermore, GATA3 expression phenocopied ECHS1 in predicting ccRCC progression and patient survival. The AMPK-GATA3-ECHS1 pathway may offer new therapeutic approaches and prognostic assessment for ccRCC in the clinic. SIGNIFICANCE: These findings uncover molecular mechanisms underlying lipid accumulation in ccRCC, suggesting the AMPK-GATA3-ECHS1 pathway as a potential therapeutic target and prognostic biomarker.
Assuntos
Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Lipogênese/genética , Transdução de Sinais/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminoácidos de Cadeia Ramificada/análise , Aminoácidos de Cadeia Ramificada/biossíntese , Animais , Carcinogênese/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Regulação para Baixo , Enoil-CoA Hidratase/metabolismo , Ácidos Graxos/análise , Ácidos Graxos/biossíntese , Feminino , Fator de Transcrição GATA3/metabolismo , Células HEK293 , Humanos , Rim/patologia , Rim/cirurgia , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Knockout , Pessoa de Meia-Idade , Nefrectomia , Prognóstico , Intervalo Livre de Progressão , Adulto JovemRESUMO
Branched-chain amino acids (BCAAs) are essential amino acids, controlling cellular metabolic processes as signaling molecules; therefore, utilization of intracellular BCAAs may be regulated by the availability of nutrients in the environment. However, spatial and temporal regulation of intracellular BCAA concentration in response to environmental conditions has been unclear due to the lack of suitable methods for measuring BCAA concentrations inside single living cells. Here, we developed a Förster resonance energy transfer (FRET)-based genetically encoded biosensor for BCAAs, termed optical biosensor for leucine-isoleucine-valine (OLIVe). The biosensor showed approximately 2-fold changes in FRET values corresponding to BCAA concentrations. Importantly, FRET signals from HeLa cells expressing OLIVe in the cytoplasm and nucleus correlated with bulk intracellular BCAA concentrations determined from populations of cells by a biochemical method, and were decreased by knockdown of L-type amino acid transporter 1 (LAT1), a transporter for BCAAs, indicating that OLIVe can reliably report intracellular BCAA concentrations inside single living cells. We also succeeded in imaging BCAA concentrations in the mitochondria using mitochondria-targeted OLIVe. Using the BCAA imaging technique, we found apparently correlated concentrations between the cytoplasm and the mitochondria. We also found that extracellular non-BCAA amino acids affected intracellular BCAA concentrations. Of these amino acids, extracellular glutamine markedly increased intracellular BCAA concentrations in a LAT1-dependent manner. Unexpectedly, extracellular pyruvate was also found to have significant positive effects on maintaining intracellular BCAA concentrations, suggesting that the cells have pyruvate-dependent systems to import BCAAs and/or to regulate BCAA metabolism.
Assuntos
Aminoácidos de Cadeia Ramificada/análise , Técnicas Biossensoriais/métodos , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/efeitos da radiação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/efeitos da radiação , Transferência Ressonante de Energia de Fluorescência/métodos , Células HeLa , Humanos , Luz , Proteínas Luminescentes/genética , Proteínas Luminescentes/efeitos da radiação , Mitocôndrias/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/efeitos da radiaçãoRESUMO
Diabetic kidney disease is a serious complication of diabetes that can ultimately lead to end-stage renal disease. The pathogenesis of diabetic kidney disease is complex, and fundamental research is still required to provide a better understanding of the driving forces behind it. We report regional metabolic aberrations from an untargeted mass spectrometry imaging study of kidney tissue using an insulinopenic rat model of diabetes. Diabetes was induced by intravenous injection of streptozotocin, and kidneys were harvested 2 weeks thereafter. Imaging was performed using nanospray desorption electrospray ionization connected to a high-mass-resolving mass spectrometer. No histopathological changes were observed in the kidney sections; however, mass spectrometry imaging revealed a significant increase in several 18-carbon unsaturated non-esterified fatty acid species and monoacylglycerols. Notably, these 18-carbon acyl chains were also constituents of several increased diacylglycerol species. In addition, a number of short- and long-chain acylcarnitines were found to be accumulated while several amino acids were depleted. This study presents unique regional metabolic data indicating a dysregulated energy metabolism in renal mitochondria as an early response to streptozotocin-induced type I diabetes. Graphical abstract.
Assuntos
Aminoácidos de Cadeia Ramificada/análise , Carnitina/análogos & derivados , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/patologia , Rim/patologia , Lipídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Carnitina/análise , Diglicerídeos/análise , Ácidos Graxos/análise , Rim/química , Masculino , Monoglicerídeos/análise , Oxirredução , Ratos , Ratos Sprague-DawleyRESUMO
Huntington's disease (HD) is a hereditary and fatal disease causing profound neurodegeneration. Deficits in cerebral energy and neurotransmitter metabolism have been suggested to play a central role in the neuronal dysfunction and death associated with HD. The branched-chain amino acids (BCAAs), leucine, isoleucine and valine, are important for cerebral nitrogen homeostasis, neurotransmitter recycling and can be utilized as energy substrates in the tricarboxylic acid (TCA) cycle. Reduced levels of BCAAs in HD have been validated by several reports. However, it is still unknown how cerebral BCAA metabolism is regulated in HD. Here we investigate the metabolism of leucine and isoleucine in the R6/2 mouse model of HD. Acutely isolated cerebral cortical and striatal slices of control and R6/2 mice were incubated in media containing 15N- or 13C-labeled leucine or isoleucine and slice extracts were analyzed by gas chromatography-mass spectrometry (GC-MS) to determine isotopic enrichment of derived metabolites. Elevated BCAA transamination was found from incubations with [15N]leucine and [15N]isoleucine, in both cerebral cortical and striatal slices of R6/2 mice compared to controls. Metabolism of [U-13C]leucine and [U-13C]isoleucine, entering oxidative metabolism as acetyl CoA, was maintained in R6/2 mice. However, metabolism of [U-13C]isoleucine, entering the TCA cycle as succinyl CoA, was elevated in both cerebral cortical and striatal slices of R6/2 mice, suggesting enhanced metabolic flux via this anaplerotic pathway. To support the metabolic studies, expression of enzymes in the BCAA metabolic pathway was assessed from a proteomic resource. Several enzymes related to BCAA metabolism were found to exhibit augmented expression in the R6/2 brain, particularly related to isoleucine metabolism, suggesting an increase in the BCAA metabolic machinery. Our results show that the capacity for cerebral BCAA metabolism, predominantly of isoleucine, is amplified in the R6/2 brain and indicates that perturbations in cerebral BCAA homeostasis could have functional consequences for HD pathology.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Encéfalo/metabolismo , Doença de Huntington/metabolismo , Aminoácidos de Cadeia Ramificada/análise , Animais , Encéfalo/patologia , Química Encefálica , Modelos Animais de Doenças , Doença de Huntington/patologia , Masculino , Redes e Vias Metabólicas , CamundongosRESUMO
Objectives: An important aspect of bipolar disorder (BD) research is the identification of biomarkers pertaining to the somatic health state. The branched-chain essential amino acids (BCAAs), viz valine, leucine and isoleucine, have been proposed as biomarkers of an individual's health state, given their influence on protein synthesis and gluconeogenesis inhibition.Methods: BCAA levels of 141 euthymic/subsyndromal individuals with BD and 141 matched healthy controls (HC) were analysed by high-pressure lipid chromatography and correlated with clinical psychiatric, anthropometric and metabolic parameters.Results: BD and HC did not differ in valine and isoleucine, whereas leucine was significantly lower in BD. Furthermore, correlations were found between BCAAs and anthropometric and glucose metabolism data. All BCAAs correlated with lipid metabolism parameters in females. There were no associations between BCAAs and long-term clinical parameters of BD. A negative correlation was found between valine and Hamilton Depression-Scale, and Beck Depression Inventory II, in male individualsConclusions: Our results indicate the utility of BCAAs as biomarkers for the current state of health, also in BD. As BD individuals have a high risk for overweight/obesity, in association with comorbid medical conditions (e.g. cardiovascular diseases or insulin resistance), health state markers are urgently required. However, no illness-specific associations were found in this euthymic/subsyndromal BD group.
Assuntos
Aminoácidos de Cadeia Ramificada/análise , Biomarcadores/análise , Transtorno Bipolar/diagnóstico , Transtorno Bipolar/metabolismo , Adulto , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Comorbidade , Metabolismo Energético , Feminino , Humanos , Isoleucina/análise , Leucina/análise , Masculino , Pessoa de Meia-Idade , Obesidade/metabolismo , Fatores Sexuais , Valina/análiseRESUMO
Metabolism of human adipose tissue during acute exercise is monitored by microdialysis. The metabolites are analysed in the microdialysate using capillary electrophoresis (CE) on a short separation path in combination with a contactless conductivity detector (C4D). Four completely new CE/C4D methods were developed for determination of nutrients and metabolites in 10⯵L of microdialysate. All methods are characterised by a short separation time and simple sample preparation based primarily on 4-fold dilution of microdialysate. The intra-day repeatability for the migration time varied in the range 0.4 - 0.9% and that for the peak area equalled 0.7 - 2.4%; the inter-day repeatability of the migration time was in the range 1.2 - 2.3% and the range for the peak area was 2.5 - 5.0%; all the values were measured as RSD. The developed determination was employed for sequenced monitoring of the levels of lactate, glycerol and branched chain amino acids in microdialysates taken from the abdominal adipose tissue during acute physical exercise. The stress test lasted 3â¯h and the metabolites were monitored at 15â¯min intervals.
Assuntos
Tecido Adiposo/metabolismo , Eletroforese Capilar/métodos , Metabolômica/métodos , Microdiálise/métodos , Adulto , Aminoácidos de Cadeia Ramificada/análise , Condutividade Elétrica , Exercício Físico/fisiologia , Glicerol/análise , Humanos , Ácido Láctico/análise , MasculinoRESUMO
Branched-chained amino acids (BCAA) are essential dietary components for humans and can act as potential biomarkers for diabetes development. To efficiently estimate dietary intake, we developed a BCAA database for 1331 food items found in the French Centre d'Information sur la Qualité des Aliments (CIQUAL) food table by compiling BCAA content from international tables, published measurements, or by food similarity as well as by calculating 267 items from Greek, Turkish, Romanian, and Moroccan mixed dishes. The database embedded in MEDIPAD software capable of registering 24 h of dietary recalls (24HDR) with clinical and genetic data was evaluated based on archived 24HDR of the Saint Pierre Institute (France) from 2957 subjects, which indicated a BCAA content up to 4.2 g/100 g of food and differences among normal weight and obese subjects across BCAA quartiles. We also evaluated the database of 119 interviews of Romanians, Turkish and Albanians in Greece (27â»65 years) during the MEDIGENE program, which indicated mean BCAA intake of 13.84 and 12.91 g/day in males and females, respectively, comparable to other studies. The MEDIPAD is user-friendly, multilingual, and secure software and with the BCAA database is suitable for conducting nutritional assessment in the Mediterranean area with particular facilities for food administration.
Assuntos
Aminoácidos de Cadeia Ramificada/análise , Bases de Dados Factuais/estatística & dados numéricos , Análise de Alimentos/métodos , Avaliação Nutricional , Software , Adulto , Idoso , Feminino , França , Grécia , Humanos , Masculino , Região do Mediterrâneo , Pessoa de Meia-Idade , Inquéritos NutricionaisRESUMO
The effect of adding amino acids on wine aroma is largely influenced by nutritional status of grape must. In this study, the effects of linoleic acid (LA) content on the aromatic function of branched-chain amino acids (BCAAs) addition were investigated in alcoholic fermentation of Cabernet Sauvignon wine. The results showed that initial LA content in must significantly influenced the effect of BCAAs addition on volatiles in final wine. Adding BCAAs (140â¯mg/L of l-leucine, 117â¯mg/L of l-isoleucine and 118â¯mg/L of l-valine) in must with low LA content (12â¯mg/L) promoted the production of most volatiles, including higher alcohols (isobutanol, 2-phenylethanol), fatty acids (hexanoic acid, octanoic acid, decanoic acid) and esters (ethyl acetate, isoamyl acetate, 2-phenethyl acetate and ethyl octanoate), which were well consistent with previous literatures. However, this function disappeared or even became inhibition with increasing LA content in must, especially in 120â¯mg/L LA must, the total contents of higher alcohol, acetate esters and ethyl esters were 33.9%, 18.1% and 54.2% lower than those in the control without BCAAs addition, respectively. The transcriptional data revealed that several major genes including GAP1, ADH1, ATF1, ACC1, FAS1 and OLE1 were marked repressed by high LA content. Our data indicated that LA can regulate the expressions of related functional genes to efficiently influence the formations of volatiles in BCAAs supplemented wines. Therefore, it is essential to consider initial content of unsaturated fatty acids (LA) in must when using the strategy that supplying amino acids (BCAAs) to modulate aromatic quality of wines.
Assuntos
Aminoácidos de Cadeia Ramificada/análise , Ácido Linoleico/análise , Odorantes/análise , Vitis/química , Vinho/análise , Aminoácidos de Cadeia Ramificada/metabolismo , Fermentação , Saccharomyces cerevisiae/metabolismo , Compostos Orgânicos Voláteis/análiseRESUMO
NMR studies of large proteins are complicated by pronounced spectral overlap and large line width. Reducing complexity by [13C, 1H] selective labeling of l-Val, l-Leu, and/or l-Ile residues in combination with optional perdeuteration is therefore commonly approached by supplying labeled amino acid precursors into bacterial expression cultures, although often compromised by high label costs, precursor instability, and label scrambling. Cell-free expression combines efficient production of membrane proteins with significant advantages for protein labeling such as small reaction volumes, defined amino acid pools, and reliable label incorporation. While amino acid specific isotopic labeling of proteins is routine application, the amino acid methyl side-chain labeling was so far difficult as appropriately labeled amino acids are hardly available. On the basis of recent proteome analyses of cell-free lysates, we have developed a competitive strategy for efficient methyl labeling of proteins based on conversion of supplied precursors. Pathway complexity of methyl side-chain labeling was reduced by implementing the promiscuous aminotransferase IlvE catalyzing the selective l-Leu, l-Val, or l-Ile biosynthesis from specific ketoacid precursors. Precursor-based l-Leu and l-Val synthesis was demonstrated with the cell-free labeling of peptidyl-prolyl cis/trans isomerase cyclophilin D and of the proton pump proteorhodopsin. The strategy is fast and cost-effective and enables the straightforward methyl side-chain labeling of individual amino acid types. It can easily be applied to any cell-free synthesized protein.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Biossíntese de Proteínas , Acetolactato Sintase/metabolismo , Aminoácidos de Cadeia Ramificada/análise , Sistema Livre de Células/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Marcação por Isótopo/métodos , Metilação , Modelos MolecularesRESUMO
Sourdough fermentation by lactic acid bacteria is commonly used in bread baking, affecting several attributes of the final product. We analyzed whole-grain wheat and rye breads and doughs prepared with baker's yeast or a sourdough starter including Candida milleri, Lactobacillus brevis and Lactobacillus plantarum using non-targeted metabolic profiling utilizing LC-QTOF-MS. The aim was to determine the fermentation-induced changes in metabolites potentially contributing to the health-promoting properties of whole-grain wheat and rye. Overall, we identified 118 compounds with significantly increased levels in sourdough, including branched-chain amino acids (BCAAs) and their metabolites, small peptides with high proportion of BCAAs, microbial metabolites of phenolic acids and several other potentially bioactive compounds. We also identified 69 compounds with significantly decreased levels, including phenolic acid precursors, nucleosides, and nucleobases. Intensive sourdough fermentation had a higher impact on the metabolite profile of whole-grain rye compared to milder whole-grain wheat sourdough fermentation. We hypothesize that the increased amount of BCAAs and potentially bioactive small peptides may contribute to the insulin response of rye bread, and in more general, the overall protective effect against T2DM and CVD.