RESUMO
The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on 19-23 June 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 2) covers the recommendations on Biomarkers, IVD/CDx, LBA and Cell-Based Assays. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 9 and 7 (2024), respectively.
Assuntos
Biomarcadores , Terapia Baseada em Transplante de Células e Tecidos , Vacinas , Humanos , Biomarcadores/análise , Vacinas/imunologia , Citometria de Fluxo , Bioensaio/métodos , União Europeia , BrancosRESUMO
In recent years, an increase in the discovery and development of biotherapeutics employing new modalities, such as bioconjugates or novel routes of delivery, has created bioanalytical challenges. The inherent complexity of conjugated molecular structures means that quantification of the bioconjugate and its multiple components is critical for preclinical/clinical studies to inform drug discovery and development. Moreover, bioconjugates involve additional multifactorial complexity because of the potential for in vivo catabolism and biotransformation, which may require thorough investigations in multiple biological matrices. Furthermore, excipients that enhance absorption are frequently evaluated and employed for the development of oral and inhaled biotherapeutics. Risk-benefit assessments are required for novel or existing excipients that utilize dosages above previously approved levels. Bioanalytical methods that can measure both excipients and potential drug metabolites in biological matrices are highly relevant to these emerging bioanalysis challenges. We discuss the bioanalytical strategies for analyzing bioconjugates such as antibody-drug conjugates and antibody-oligonucleotide conjugates and review recent advances in bioanalytical methods for the quantification and characterization of novel bioconjugates. We also discuss bioanalytical considerations for both biotherapeutics and excipients through novel administration routes and review analyses in various biological matrices, from the extensively studied serum or plasma to tissue biopsy in the context of preclinical and clinical studies from both technical and regulatory perspectives.
Assuntos
Excipientes , Imunoconjugados , Descoberta de Drogas , Humanos , Imunoconjugados/uso terapêutico , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/metabolismoRESUMO
The history of humankind is marked by the constant adoption of new dietary habits affecting human physiology, metabolism, and even the development of nutrition-related disorders. Despite clear archaeological evidence for the shift from hunter-gatherer lifestyle to agriculture in Neolithic Europe [1], very little information exists on the daily dietary habits of our ancestors. By undertaking a complementary -omics approach combined with microscopy, we analyzed the stomach content of the Iceman, a 5,300-year-old European glacier mummy [2, 3]. He seems to have had a remarkably high proportion of fat in his diet, supplemented with fresh or dried wild meat, cereals, and traces of toxic bracken. Our multipronged approach provides unprecedented analytical depth, deciphering the nutritional habit, meal composition, and food-processing methods of this Copper Age individual.
Assuntos
Dieta/história , Múmias , Arqueologia , Áustria , Gorduras na Dieta , Grão Comestível , História Antiga , Humanos , Itália , Masculino , CarneAssuntos
Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/etiologia , Galantamina/uso terapêutico , Cinurenina/metabolismo , Memantina/uso terapêutico , Nootrópicos/uso terapêutico , Esquizofrenia/complicações , Adolescente , Adulto , Quimioterapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Estudos Retrospectivos , Transdução de Sinais/efeitos dos fármacos , Adulto JovemRESUMO
Novel metabolites distinct from canonical pathways can be identified through the integration of three cheminformatics tools: BinVestigate, which queries the BinBase gas chromatography-mass spectrometry (GC-MS) metabolome database to match unknowns with biological metadata across over 110,000 samples; MS-DIAL 2.0, a software tool for chromatographic deconvolution of high-resolution GC-MS or liquid chromatography-mass spectrometry (LC-MS); and MS-FINDER 2.0, a structure-elucidation program that uses a combination of 14 metabolome databases in addition to an enzyme promiscuity library. We showcase our workflow by annotating N-methyl-uridine monophosphate (UMP), lysomonogalactosyl-monopalmitin, N-methylalanine, and two propofol derivatives.
Assuntos
Proteínas Sanguíneas/metabolismo , Biologia Computacional/métodos , Bases de Dados Factuais , Cromatografia Gasosa-Espectrometria de Massas/métodos , Metaboloma , Metabolômica/métodos , Software , Bactérias/metabolismo , Cromatografia Líquida , Fezes/química , HumanosRESUMO
The AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK ß1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminopiridinas/farmacologia , Ativadores de Enzimas/farmacologia , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Indóis/farmacologia , Aminopiridinas/uso terapêutico , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/uso terapêutico , Feminino , Hipoglicemiantes/uso terapêutico , Indóis/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismoRESUMO
Obese, insulin-resistant states are characterized by a paradoxical pathogenic condition in which the liver appears to be selectively insulin resistant. Specifically, insulin fails to suppress glucose production, yet successfully stimulates de novo lipogenesis. The mechanisms underlying this dysregulation remain controversial. Here, we hypothesized that carbohydrate-responsive element-binding protein (ChREBP), a transcriptional activator of glycolytic and lipogenic genes, plays a central role in this paradox. Administration of fructose increased hepatic hexose-phosphate levels, activated ChREBP, and caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, and hepatic steatosis in mice. Activation of ChREBP was required for the increased expression of glycolytic and lipogenic genes as well as glucose-6-phosphatase (G6pc) that was associated with the effects of fructose administration. We found that fructose-induced G6PC activity is a major determinant of hepatic glucose production and reduces hepatic glucose-6-phosphate levels to complete a homeostatic loop. Moreover, fructose activated ChREBP and induced G6pc in the absence of Foxo1a, indicating that carbohydrate-induced activation of ChREBP and G6PC dominates over the suppressive effects of insulin to enhance glucose production. This ChREBP/G6PC signaling axis is conserved in humans. Together, these findings support a carbohydrate-mediated, ChREBP-driven mechanism that contributes to hepatic insulin resistance.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Frutose/toxicidade , Glucose/biossíntese , Resistência à Insulina , Insulina/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Feminino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Glucose/genética , Intolerância à Glucose/induzido quimicamente , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Insulina/genética , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled (13)C6 glucose, (13)C3 lactate, and (13)C5 glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture.
Assuntos
Isótopos de Carbono/química , Cromatografia Líquida , Hepatócitos/metabolismo , Espectrometria de Massas , Sondas Moleculares/química , Animais , Isótopos de Carbono/análise , Células Cultivadas , Glicólise , Metaboloma/fisiologia , RatosRESUMO
High fructose consumption has been implicated with deleterious effects on human health, including hyperlipidemia elicited through de novo lipogenesis. However, more global effects of fructose on cellular metabolism have not been elucidated. In order to explore the metabolic impact of fructose-containing nutrients, we applied both GC-TOF and HILIC-QTOF mass spectrometry metabolomic strategies using extracts from cultured HepG2 cells exposed to fructose, glucose, or fructose + glucose. Cellular responses were analyzed in a time-dependent manner, incubated in media containing 5.5 mM glucose + 5.0 mM fructose in comparison to controls incubated in media containing either 5.5 mM glucose or 10.5 mM glucose. Mass spectrometry identified 156 unique known metabolites and a large number of unknown compounds, which revealed metabolite changes due to both utilization of fructose and high-carbohydrate loads independent of hexose structure. Fructose was shown to be partially converted to sorbitol, and generated higher levels of fructose-1-phosphate as a precursor for glycolytic intermediates. Differentially regulated ratios of 3-phosphoglycerate to serine pathway intermediates in high fructose media indicated a diversion of carbon backbones away from energy metabolism. Additionally, high fructose conditions changed levels of complex lipids toward phosphatidylethanolamines. Patterns of acylcarnitines in response to high hexose exposure (10.5 mM glucose or glucose/fructose combination) suggested a reduction in mitochondrial beta-oxidation.
RESUMO
NADH and NADPH undergo spontaneous and enzymatic reactions that produce R and S forms of NAD(P)H hydrates [NAD(P)HX], which are not electron donors and inhibit various dehydrogenases. In bacteria, yeast (Saccharomyces cerevisiae), and mammals, these hydrates are repaired by the tandem action of an ADP- or ATP-dependent dehydratase that converts (S)-NAD(P)HX to NAD(P)H and an epimerase that facilitates interconversion of the R and S forms. Plants have homologs of both enzymes, the epimerase homolog being fused to the vitamin B6 salvage enzyme pyridoxine 5'-phosphate oxidase. Recombinant maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) NAD(P)HX dehydratases (GRMZM5G840928, At5g19150) were able to reconvert (S)-NAD(P)HX to NAD(P)H in an ATP-dependent manner. Recombinant maize and Arabidopsis epimerases (GRMZM2G061988, At5g49970) rapidly interconverted (R)- and (S)-NAD(P)HX, as did a truncated form of the Arabidopsis epimerase lacking the pyridoxine 5'-phosphate oxidase domain. All plant NAD(P)HX dehydratase and epimerase sequences examined had predicted organellar targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. In vitro transcription/translation assays confirmed that both start sites were used. Dual import assays with purified pea (Pisum sativum) chloroplasts and mitochondria, and subcellular localization of GFP fusion constructs in tobacco (Nicotiana tabacum) suspension cells, indicated mitochondrial, plastidial, and cytosolic localization of the Arabidopsis epimerase and dehydratase. Ablation of the Arabidopsis dehydratase gene raised seedling levels of all NADHX forms by 20- to 40-fold, and levels of one NADPHX form by 10- to 30-fold. We conclude that plants have a canonical two-enzyme NAD(P)HX repair system that is directed to three subcellular compartments via the use of alternative translation start sites.
Assuntos
Arabidopsis/metabolismo , NADP/metabolismo , Água/metabolismo , Zea mays/metabolismo , Arabidopsis/enzimologia , Técnicas de Inativação de Genes , Hidroliases/metabolismo , Cinética , Proteínas de Plantas/metabolismo , Estrutura Terciária de Proteína , Piridoxaminafosfato Oxidase/química , Racemases e Epimerases/química , Racemases e Epimerases/metabolismo , Homologia de Sequência do Ácido Nucleico , Frações Subcelulares/enzimologia , Zea mays/enzimologiaRESUMO
Novel plasma metabolite patterns reflective of improved metabolic health (insulin sensitivity, fitness, reduced body weight) were identified before and after a 14-17 wk weight loss and exercise intervention in sedentary, obese insulin-resistant women. To control for potential confounding effects of diet- or microbiome-derived molecules on the systemic metabolome, sampling was during a tightly-controlled feeding test week paradigm. Pairwise and multivariate analysis revealed intervention- and insulin-sensitivity associated: (1) Changes in plasma xeno-metabolites ("non-self" metabolites of dietary or gut microbial origin) following an oral glucose tolerance test (e.g. higher post-OGTT propane-1,2,3-tricarboxylate [tricarballylic acid]) or in the overnight-fasted state (e.g., lower γ-tocopherol); (2) Increased indices of saturated very long chain fatty acid elongation capacity; (3) Increased post-OGTT α-ketoglutaric acid (α-KG), fasting α-KG inversely correlated with Matsuda index, and altered patterns of malate, pyruvate and glutamine hypothesized to stem from improved mitochondrial efficiency and more robust oxidation of glucose. The results support a working model in which improved metabolic health modifies host metabolism in parallel with altering systemic exposure to xeno-metabolites. This highlights that interpretations regarding the origins of peripheral blood or urinary "signatures" of insulin resistance and metabolic health must consider the potentially important contribution of gut-derived metabolites toward the host's metabolome.
Assuntos
Trato Gastrointestinal/metabolismo , Saúde , Metaboloma , Xenobióticos/metabolismo , Adulto , Área Sob a Curva , Dieta , Análise Discriminante , Jejum/sangue , Ácidos Graxos/sangue , Feminino , Glucose/metabolismo , Teste de Tolerância a Glucose , Humanos , Análise dos Mínimos Quadrados , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/metabolismo , Fenótipo , Aptidão Física , Comportamento Sedentário , Redução de PesoRESUMO
Current tandem mass spectral libraries for lipid annotations in metabolomics are limited in size and diversity. We provide a freely available computer-generated tandem mass spectral library of 212,516 spectra covering 119,200 compounds from 26 lipid compound classes, including phospholipids, glycerolipids, bacterial lipoglycans and plant glycolipids. We show platform independence by using tandem mass spectra from 40 different mass spectrometer types including low-resolution and high-resolution instruments.
Assuntos
Bases de Dados Factuais , Lipídeos/análise , Espectrometria de Massas em Tandem/métodos , Metabolômica/métodosRESUMO
Exercise substantially improves metabolic health, making the elicited mechanisms important targets for novel therapeutic strategies. Uncoupling protein 3 (UCP3) is a mitochondrial inner membrane protein highly selectively expressed in skeletal muscle. Here we report that moderate UCP3 overexpression (roughly 3-fold) in muscles of UCP3 transgenic (UCP3 Tg) mice acts as an exercise mimetic in many ways. UCP3 overexpression increased spontaneous activity (â¼40%) and energy expenditure (â¼5-10%) and decreased oxidative stress (â¼15-20%), similar to exercise training in wild-type (WT) mice. The increase in complete fatty acid oxidation (FAO; â¼30% for WT and â¼70% for UCP3 Tg) and energy expenditure (â¼8% for WT and 15% for UCP3 Tg) in response to endurance training was higher in UCP3 Tg than in WT mice, showing an additive effect of UCP3 and endurance training on these two parameters. Moreover, increases in circulating short-chain acylcarnitines in response to acute exercise in untrained WT mice were absent with training or in UCP3 Tg mice. UCP3 overexpression had the same effect as training in decreasing long-chain acylcarnitines. Outcomes coincided with a reduction in muscle carnitine acetyltransferase activity that catalyzes the formation of acylcarnitines. Overall, results are consistent with the conclusions that circulating acylcarnitines could be used as a marker of incomplete muscle FAO and that UCP3 is a potential target for the treatment of prevalent metabolic diseases in which muscle FAO is affected.
Assuntos
Regulação da Expressão Gênica/fisiologia , Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , Resistência Física , Animais , Biomarcadores , Ingestão de Alimentos , Metabolismo Energético , Canais Iônicos/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais/genética , Músculo Esquelético/metabolismo , Oxirredução , Estresse Oxidativo , Condicionamento Físico Animal , Proteína Desacopladora 3RESUMO
Induced pluripotent stem cells are different from embryonic stem cells as shown by epigenetic and genomics analyses. Depending on cell types and culture conditions, such genetic alterations can lead to different metabolic phenotypes which may impact replication rates, membrane properties and cell differentiation. We here applied a comprehensive metabolomics strategy incorporating nanoelectrospray ion trap mass spectrometry (MS), gas chromatography-time of flight MS, and hydrophilic interaction- and reversed phase-liquid chromatography-quadrupole time-of-flight MS to examine the metabolome of induced pluripotent stem cells (iPSCs) compared to parental fibroblasts as well as to reference embryonic stem cells (ESCs). With over 250 identified metabolites and a range of structurally unknown compounds, quantitative and statistical metabolome data were mapped onto a metabolite networks describing the metabolic state of iPSCs relative to other cell types. Overall iPSCs exhibited a striking shift metabolically away from parental fibroblasts and toward ESCs, suggestive of near complete metabolic reprogramming. Differences between pluripotent cell types were not observed in carbohydrate or hydroxyl acid metabolism, pentose phosphate pathway metabolites, or free fatty acids. However, significant differences between iPSCs and ESCs were evident in phosphatidylcholine and phosphatidylethanolamine lipid structures, essential and non-essential amino acids, and metabolites involved in polyamine biosynthesis. Together our findings demonstrate that during cellular reprogramming, the metabolome of fibroblasts is also reprogrammed to take on an ESC-like profile, but there are select unique differences apparent in iPSCs. The identified metabolomics signatures of iPSCs and ESCs may have important implications for functional regulation of maintenance and induction of pluripotency.
Assuntos
Células-Tronco Embrionárias/metabolismo , Metabolômica , Fosfatidilcolinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Aminoácidos/metabolismo , Animais , Regulação para Baixo , Cromatografia Gasosa-Espectrometria de Massas , CamundongosRESUMO
Lipid secretions from algae pose a great opportunity for engineering biofueler feedstocks. The lipid exudates could be interesting from a process engineering perspective because lipids could be collected directly from the medium without harvesting and disrupting cells. We here report on the extracellular secretions of algal metabolites from the strain UTEX 2341 (Chlorella minutissima) into the culture medium. No detailed analysis of these lipid secretions has been performed to date. Using multiple mass spectrometric platforms, we observed around 1000 compounds and were able to annotate 50 lipids by means of liquid chromatography coupled to accurate mass quadrupole time-of-flight mass spectrometry (LC-QTOF), direct infusion with positive and negative electrospray ion trap mass spectrometry and gas chromatography coupled to mass spectrometry (GC-MS). These compounds were annotated by tandem mass spectral (MS/MS) database matching and retention time range filtering. We observed a series of triacylglycerols (TG), sulfoquinovosyldiacylglycerols (SQDG), phosphatidylinositols and phosphatidylglycerols, as well as betaine lipids diacylglyceryl-N,N,N-trimethylhomoserines (DGTS).
Assuntos
Chlorella/química , Cromatografia Líquida/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Lipídeos/análise , Espectrometria de Massas em Tandem/métodos , Biocombustíveis , Chlorella/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Metabolismo dos Lipídeos , Lipídeos/químicaRESUMO
Consumption of large amounts of fructose or sucrose increases lipogenesis and circulating triglycerides in humans. Although the underlying molecular mechanisms responsible for this effect are not completely understood, it is possible that as reported for rodents, high fructose exposure increases expression of the lipogenic enzymes fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC-1) in human liver. Since activation of the hexosamine biosynthesis pathway (HBP) is associated with increases in the expression of FAS and ACC-1, it raises the possibility that HBP-related metabolites would contribute to any increase in hepatic expression of these enzymes following fructose exposure. Thus, we compared lipogenic gene expression in human-derived HepG2 cells after incubation in culture medium containing glucose alone or glucose plus 5 mM fructose, using the HBP precursor 10 mM glucosamine (GlcN) as a positive control. Cellular metabolite profiling was conducted to analyze differences between glucose and fructose metabolism. Despite evidence for the active uptake and metabolism of fructose by HepG2 cells, expression of FAS or ACC-1 did not increase in these cells compared with those incubated with glucose alone. Levels of UDP-N-acetylglucosamine (UDP-GlcNAc), the end-product of the HBP, did not differ significantly between the glucose and fructose conditions. Exposure to 10 mM GlcN for 10 minutes to 24 hours resulted in 8-fold elevated levels of intracellular UDP-GlcNAc (P<0.001), as well as a 74-126% increase in FAS (P<0.05) and 49-95% increase in ACC-1 (P<0.01) expression above controls. It is concluded that in HepG2 liver cells cultured under standard conditions, sustained exposure to fructose does not result in an activation of the HBP or increased lipogenic gene expression. Should this scenario manifest in human liver in vivo, it would suggest that high fructose consumption promotes triglyceride synthesis primarily through its action to provide lipid precursor carbon and not by activating lipogenic gene expression.
Assuntos
Frutose/farmacologia , Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Acetil-CoA Carboxilase/metabolismo , Ácido Graxo Sintases/metabolismo , Frutose/metabolismo , Glucosamina/farmacologia , Glucose/metabolismo , Células Hep G2 , Humanos , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
We describe the resolution of heparan sulfate (HS) isomers by chromatographic methods and their subsequent differentiation by mass spectrometry (MS), ion mobility, and (1)H nuclear magnetic resonance (NMR) analysis. The two purified hexasaccharide isomers produced nearly identical MS spectra, quantitative disaccharide profiles, and partial enzymatic digestions. However, both tandem spectrometry (MS(2)) and ion mobility spectrometry (IMS) indicated structural differences existed. All data suggested the distinction between the two hexasaccharides resided in their uronic acid stereochemistries. Glucuronic (GlcA) and iduronic acids (IdoA) were subsequently defined by (1)H NMR analysis completing the structural analysis and verifying the unique structures initially indicated by MS(2) and IMS. Our results suggest that IMS may be a powerful tool in the rapid differentiation of GlcA and IdoA containing isomers in the absence of prior structural knowledge.
Assuntos
Ácido Glucurônico/química , Heparitina Sulfato/isolamento & purificação , Ácido Idurônico/química , Oligossacarídeos/isolamento & purificação , Sequência de Carboidratos , Heparitina Sulfato/química , Isomerismo , Espectroscopia de Ressonância Magnética , Oligossacarídeos/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The presence of 3-O-sulfated glucosamine residues in heparin or heparan sulfate plays a role in binding to antithrombin III and HSV infection. In this study, tandem mass spectrometry was used to differentiate between two heparin disaccharide isomers containing variable sulfate at C6 in a common disaccharide and C3 in a more rare one. The dissociation patterns shown by MS(2) and MS(3) were clearly distinguishable between the isomers, allowing their differentiation and quantitation. Using this technique, we show that an octasaccharide with 11 sulfate groups with high affinity for inflammatory chemokine CCL2 does not contain 3-O-sulfated disaccharides.