RESUMEN
Oxalate and citrate in 24 h urine and serum are considered to be associated with the incidence and recurrence risk of calcium oxalate kidney stones. The quantification of oxalate and citrate contributes to understand the pathological metabolism of kidney stones and guide the early diagnosis and recurrence monitoring. Although simultaneous quantification of oxalate and citrate in urine using liquid chromatography tandem mass spectrometry (LC-MS/MS) have been reported, the optimization of chromatographic column, mobile phase and mass spectrometry (MS) parameters has not been performed. In addition, these is a lack of suitable method for simultaneous detection of oxalate and citrate both in serum and urine. Therefore, we developed a method for the simultaneous determination of oxalate and citrate in urine and serum based on ion-pairing reversed-phase (IP-RP) LC-MS/MS. Herein, five ion-pair reagents, namely, triethanolamine, dimethylbutyl amine, diisopropenyl amine, N,N-dimethylcyclohexylamine and tripropylamine, and three ion-pairing reagent (IPR) buffers, namely, acetic acid, hexafluoro-2-isopropanol, and hexafluoro-2-methyl-2-propanol, were compared in regard to their chromatographic peak abundance and separation of oxalate and citrate. Moreover, MS parameters and the multiple reaction monitoring (MRM) conditions were also evaluated and optimized to obtain the maximum peak abundance. After that, the method was validated in the linear range of 0.25-1000 µM, and the correlation coefficient was ≥ 0.99. The precision and accuracy were < 14.70% and < 19.73%, respectively. The extraction recovery was 80.53-108.79%, and the matrix effect was < 8.79%. The quality control samples were stable at room temperature for 4 h, 4 °C for 24 h, and for 3 freeze-thaw cycles. Finally, this method was applied to the determination of oxalate and citrate in the serum and urine of rats with calcium oxalate kidney stones. The establishment of a stable and effective oxalate and citrate detection method is conducive to the diagnosis and monitoring of kidney stones.
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Cálculos Renales , Espectrometría de Masas en Tándem , Aminas , Animales , Oxalato de Calcio , Cromatografía Liquida/métodos , Ácido Cítrico , Iones , Cálculos Renales/diagnóstico , Nefrolitiasis , Oxalatos , Ratas , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Non-small-cell lung cancer (NSCLC) is one of the main types of lung cancer. Due to lack of effective biomarkers for early detection of NSCLC, the therapeutic effect is not ideal. This study aims to reveal potential biomarkers for clinical diagnosis. METHODS: The plasma metabolic profiles of the patients were characterized by liquid chromatography-mass spectrometry (LC-MS). Differential metabolites were screened by p less than 0.05 and VIP greater than 1. Multivariate statistical analysis was used to search for potential biomarkers. Receiver operating characteristic (ROC) curve was used to evaluate the predictors of potential biomarkers. Pathway enrichment analysis was performed on metabolomics data by Ingenuity Pathway Analysis (IPA) and transcriptomics data from GEO were used for validation. RESULTS: A plasma metabolite biomarker panel including 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE) and arachidonic acid was chose. The area under the ROC curve were 0.917, 0.900 and 0.867 for the panel in the different algorithm like Partial Least Squares Discrimination Analysis (PLS-DA), Support Vector Machine (SVM), Random Forest (RF). The candidate biomarkers were associated with the Akt pathway. Genes involved in the biological pathway had significant changes in the expression levels. CONCLUSION: 13(S)-HODE and arachidonic acid may be potential biomarkers of NSCLC. The Akt pathway was associated with this biomarker panel in NSCLC. Further studies are needed to clarify the mechanisms of disruption in this pathway.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Ácido Araquidónico , Biomarcadores/metabolismo , Biomarcadores de Tumor , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Proliferación Celular , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Metabolómica/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Curva ROC , TranscriptomaRESUMEN
RATIONALE: The mechanism of lipid metabolism disorder in type 2 diabetes (T2DM) remains unclear. This study aimed to reveal the mechanism underlying dysregulated lipid metabolism in T2DM through bile acid metabolism. METHODS: A db/db mouse model was employed to investigate the alteration of bile acid profiles in T2DM. Ultrahigh-performance liquid chromatography with tandem mass spectrometry was used to quantify the detailed bile acid levels in each compartment of enterohepatic circulation. The pathological change of mouse liver was assessed by liver histology and serum biochemical assays. The expression level of bile acid-related transporters and synthases was measured with Western blot analysis. RESULTS: The results showed that T2DM can result in severe liver fat accumulation and liver damage. In addition, compared to the control group, in T2DM mice, bile acid synthesis is reduced, while the level of bile acids is increased at the storage sites and the reabsorption sites, but there are subtle gender differences. Further, the ratio of conjugated bile acids in total bile acid in the liver of T2DM mice increased significantly relative to the control group for both female and male mice. CONCLUSIONS: In T2DM, bile acid metabolism is disordered in both male and female mice, which could be the underlying mechanism of dysregulated lipid metabolism in T2DM.
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Ácidos y Sales Biliares/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo de los Lípidos , Animales , Ácidos y Sales Biliares/química , Cromatografía Líquida de Alta Presión , Circulación Enterohepática , Grasas/química , Grasas/metabolismo , Femenino , Humanos , Hígado/química , Hígado/metabolismo , Masculino , Metabolómica , Ratones , Espectrometría de Masas en TándemRESUMEN
Rheumatoid arthritis (RA) is a common autoimmune and inflammatory disease worldwide, but understanding its pathogenesis is still limited. In this study, plasma untargeted metabolomics of a discovery cohort and targeted analysis of a verification cohort were performed by gas chromatograph mass spectrometry (GC/MS). Univariate and multivariate statistical analysis were utilized to reveal differential metabolites, followed by the construction of biomarker panel through random forest (RF) algorithm. The pathways involved in RA were enriched by differential metabolites using Ingenuity Pathway Analysis (IPA) suite. Untargeted metabolomics revealed eighteen significantly altered metabolites in RA. Among these metabolites, a three-metabolite marker panel consisting of L-cysteine, citric acid and L-glutamine was constructed, using random forest algorithm that could predict RA with high accuracy, sensitivity and specificity based on a multivariate exploratory receiver operator characteristic (ROC) curve analysis. The panel was further validated by support vector machine (SVM) and partial least squares discriminant analysis (PLS-DA) algorithms, and also verified with targeted metabolomics using a verification cohort. Additionally, the dysregulated taurine biosynthesis pathway in RA was revealed by an integrated analysis of metabolomics and transcriptomics. Our findings in this study not only provided a mechanism underlying RA pathogenesis, but also offered alternative therapeutic targets for RA.
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Artritis Reumatoide , Metabolómica , Artritis Reumatoide/diagnóstico , Biomarcadores , HumanosRESUMEN
Lung cancer (LC) is one of the most malignant cancers in the world, but currently, it lacks effective noninvasive biomarkers to assist its early diagnosis. Our study aims to discover potential serum diagnostic biomarkers for LC. In our study, untargeted serum metabolomics of a discovery cohort and targeted analysis of a test cohort were performed based on gas chromatography-mass spectrometry. Both univariate and multivariate statistical analyses were employed to screen for differential metabolites between LC and healthy control (HC), followed by the selection of candidate biomarkers through multiple algorithms. The results showed that 15 metabolites were significantly dysregulated between LC and HC, and a panel, comprising cholesterol, oleic acid, myo-inositol, 2-hydroxybutyric acid, and 4-hydroxybutyric acid, was demonstrated to have excellent differentiating capability for LC based on multiple classification modelings. In addition, the molecular interaction analysis combined with transcriptomics revealed a close correlation between the candidate biomarkers and LC proliferation via a Ca2+ signaling pathway. Our study discovered that cholesterol, oleic acid, myo-inositol, 2-hydroxybutyric acid, and 4-hydroxybutyric acid in combination could be a promising diagnostic biomarker for LC, and most importantly, our results will shed some light on the pathophysiological mechanism underlying LC to understand it deeply. The data that support the findings of this study are openly available in MetaboLights at https://www.ebi.ac.uk/metabolights/, reference number MTBLS1517.
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Neoplasias Pulmonares , Transcriptoma , Biomarcadores/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Señalización del Calcio , Proliferación Celular , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , MetabolómicaRESUMEN
The correlation among stationary phases, ion-pairing reagents (IPR) and sequences for ion-pair reversed-phase liquid chromatography mass spectrometry (IP-RP LC-MS) analysis of oligonucleotide (ODN) remains unclear. The present study aimed to evaluate such correlation using particle-packed C18 columns in order to search for the optimal combination among them. Five C18 columns packed with core-shell silica, polymer, porous silica and hybrid particles, respectively, were evaluated for the analysis of synthetic and chemically modified ODNs with six different IPRs. Our results showed that silica-based porous particles, compared to other particles, retained ODN the strongest no matter which IPR was used. Meanwhile, among the six IPRs hexylamine (HA) produced the longest retention for all ODNs, regardless of the types of C18 particles. For the separation of ODNs, C18 columns performed similarly under identical LC conditions. However, the separation ability of C18 columns is highly dependent on the type of IPR and ODN sequences. Moreover, the type of particles has little impact on the signals of ODNs for the majority of synthetic sequences, but such impact could be dramatic for chemically modified sequences. On the other hand, both the type of IPR and ODN sequence have a significant effect on MS signals for synthetic and chemically modified ODNs.
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Cromatografía Liquida/métodos , Cromatografía de Fase Inversa/métodos , Oligonucleótidos/análisis , Oligonucleótidos/química , Polímeros/química , Dióxido de Silicio/química , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Lack of efficient noninvasive biomarkers for differentiating prostate cancer (PCa) and benign prostate hyperplasia (BPH) is a serious concern for men's health worldwide. In this study, we aimed to improve the diagnostic capability of the existing noninvasive biomarkers for PCa. GC-MS-based untargeted metabolomics was employed to analyze plasma samples for 41 PCa patients and 38 BPH controls. Both univariate and multivariate statistical analyses were performed to screen for differential metabolites between PCa and BPH, followed by the selection of potential biomarkers through machine learning. The chosen candidate biomarkers were then verified by targeted analysis and transcriptome data. The results showed that twelve metabolites were significantly dysregulated between PCa and BPH, three metabolites including L-serine, myo-inositol, and decanoic acid could be potential biomarkers for discriminating PCa from BPH. Most importantly, ROC curve analysis demonstrated that the involvement of the three potential biomarkers has increased the area under the curve (AUC) value of cPSA and tPSA from 0.542 and 0.592 to 0.781, respectively. Therefore, it was concluded that the involvement of L-serine, myo-inositol, and decanoic acid can largely improve the diagnostic capability of the commonly used noninvasive biomarkers in the clinic for differentiating PCa from BPH.
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Hiperplasia Prostática , Neoplasias de la Próstata , Biomarcadores , Biomarcadores de Tumor , Diagnóstico Diferencial , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Metabolómica , Antígeno Prostático Específico , Hiperplasia Prostática/diagnóstico , Neoplasias de la Próstata/diagnósticoRESUMEN
Plasticware and glassware used in the sample processing stage could result in different analysis performance for macromolecules, which led to the speculation that a similar effect could happen to small molecules. To confirm the speculation, pooled human plasma sample spiked with and without metabolite standards was prepared with three most commonly used container materials (glass, deactivated glass and polypropylene) supplied by different manufacturers after a two-step liquid-liquid extraction, followed by gas chromatography mass spectrometry (GC-MS) based untargeted and targeted metabolomics. The results showed that both GC-MS-based targeted and untargeted metabolomics could be influenced significantly by not only the container material but also the manufacturing procedures employed by different vendors. As a general guideline, it would be highly practical to use the same type of container from the same manufacturer during a whole project. Our study could be extremely valuable for the fellow researchers when dealing with the sample containers for GC-MS-based untargeted and targeted metabolomics.
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Metabolómica , Plasma , Cromatografía de Gases y Espectrometría de Masas , Humanos , Estándares de Referencia , Manejo de EspecímenesRESUMEN
Sample preparation plays a crucial part in plasma metabolomics. In order to obtain an optimal sample extraction method for gas chromatography mass spectrometry (GC-MS)-based plasma metabolomics, five different extraction strategies including protein precipitation, liquid-liquid extraction and solid-phase extraction were evaluated systematically for both plasma untargeted- and targeted-metabolomics. The comprehensive evaluation revealed that the all-in-one sample preparation method, MeOH-MTBE-H2O (1:5:1.5, v/v/v), was the optimal extraction method for both untargeted- and targeted-metabolomics. Next, the optimal sample preparation protocol was applied in plasma metabolomics of osteoarthritis (OA). A panel containing cholesterol, lactic acid, stearic acid, alpha-tocopherol and oxalic acid was selected as candidate biomarker to distinguish OA patients from healthy controls (HC) based on the support vector machine (SVM) classification model. The discriminating capability of the candidate biomarker panel was further validated successfully with logistic regression and principal components analysis (PCA) analysis. Therefore, the panel could potentially act as diagnostic biomarker for osteoarthritis.
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Fraccionamiento Químico/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Metabolómica/métodos , Osteoartritis/sangre , Manejo de Especímenes/métodos , Anciano , Biomarcadores/sangre , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis de Componente Principal , Curva ROCRESUMEN
Sodium taurocholate cotransporting polypeptide (NTCP) is an important hepatocyte transporter, while its physiological functions require further investigation. In our study, an integrated plasma and liver GC-MS- and LC-MS-based metabolomics strategy with an optimized two-step liquid-liquid extraction was utilized to explore the physiological functions of NTCP via a knockout (KO) mouse model. The present study found that NTCP deficiency resulted in obvious metabolic change in the plasma and liver of mice. Totally, 102 and 87 differential metabolites were discovered in the liver and plasma, respectively. Pathway analysis revealed that the metabolism of tyrosine, glycine, taurine, fatty acid and glycerophospholipid as well as the biosynthesis of tryptophan, pantothenate and CoA were significantly dysregulated in the Ntcp KO mice, indicating that NTCP is closely involved in these metabolic pathways. Moreover, L-tryptophan, cadaverine and D-pantothenic acid could serve as the diagnostic biomarker for NTCP deficiency. Our study provided deep insights into the physiological functions of NTCP, and the findings would hold the great potential to be used for the discovery of new therapeutic and diagnostic strategies for NTCP deficiency clinically.
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Cromatografía Liquida/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Metaboloma/fisiología , Metabolómica/métodos , Transportadores de Anión Orgánico Sodio-Dependiente , Simportadores , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Hígado/química , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/fisiología , Simportadores/genética , Simportadores/metabolismo , Simportadores/fisiologíaRESUMEN
BACKGROUND: The role of multidrug resistance-associated protein 3 (Mrp3) in the transport of bile acid (BA) in drug-induced cholestasis has not been well studied. OBJECTIVE: In this study, wild type and Mrp3 knockout (Mrp3-/-) mice under normal physiological and lithocholic acid (LCA)-induced cholestatic conditions were employed to investigate the role of Mrp3 in BA transport. METHODS: The levels of BA in serum, liver, gallbladder, intestine, kidney, feces and urine were quantified in both wild type and Mrp3-/- mice via ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-MS/MS). Quantitative real-time PCR (RT-PCR) analysis was used to measure the expression of genes related to the transport and synthesis of BA. RESULTS: The results showed that the liver did not suffer more serious damage as a result of cholestasis when Mrp3 was depleted. The level of some individual bile acids changed apparently in the compartments of enterohepatic circulation (EHC) between the two control and model groups, respectively, but the level of serum total bile acid was only slightly reduced for Mrp3-/- groups. In addition, the level of BA-related efflux transporters and synthases increased significantly when Mrp3 was knocked out under normal physiological conditions, but a negligible alteration appeared under cholestatic conditions. CONCLUSION: Our results indicated that Mrp3 could be responsible for the transport of some specific bile acids, and part of the Mrp3 role could be compensated for by other transporters. Moreover, Mrp3 deficiency has a direct effect on the expression of BA-related synthases and efflux transporters under normal physiological conditions, but this effect could be less prominent under cholestatic conditions. This study could provide much valuable insight into the physiological function of Mrp3 in the transport of bile acids.
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Ácidos y Sales Biliares/metabolismo , Colestasis/inducido químicamente , Ácido Litocólico/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Animales , Ácidos y Sales Biliares/sangre , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismoRESUMEN
Lung cancer has surpassed breast cancer as the leading cause of cancer death in females in developed countries and the leading cause of cancer death in males. Despite extensive research on lung cancer, the pathogenesis of lung cancer is not fully understood. ALKBH1 is a 2-oxoglutarate and Fe (II)-dependent dioxygenase responsible for the demethylation of 6-methyladenine (m6A) in RNA and is essential to multiple cellular processes in human. Numerous recent studies suggest that ALKBH1 plays a role in tumorigenesis and tumor progression, but the role of ALKBH1 in lung cancer is largely unknown. In this study, we demonstrated that the expression levels of ALKBH1 in lung cancer tissues and cells were up regulated. The invasion and migration abilities of lung cancer cells were significantly suppressed in vitro upon the silencing of ALKBH1 while they were significantly promoted upon its overexpression. We next characterized the enzyme biochemically by analyzing the contribution of essential residues Y184, H231, D233, H287, R338, and R344 to its m6A demethylation activity. Lastly, our 3.1-Å crystal structure of mouse ALKBH1 revealed that the N-terminal domain of the protein forms close contacted with the core catalytic domain and might be responsible for the recognition of nucleic acid substrates. In summary, our combined cellular, biochemical, and structural results provide insight into the potential ALKBH1-based drug design for cancer therapies.
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Adenina/análogos & derivados , Histona H2a Dioxigenasa, Homólogo 1 de AlkB/biosíntesis , Desmetilación , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , ARN/metabolismo , Células A549 , Adenina/metabolismo , Histona H2a Dioxigenasa, Homólogo 1 de AlkB/genética , Secuencia de Aminoácidos , Animales , Femenino , Humanos , Neoplasias Pulmonares/genética , Ratones , Ratones Endogámicos C57BL , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN/genéticaRESUMEN
GC-MS platform has been proved to be an important analytical technique for plasma metabolomics, but lack of efficient sample preparation strategies prior to sample injection has limited its wide application. In the present work, twenty two extraction protocols including protein precipitation (PPT), liquid-liquid extraction (LLE), solid-phase extraction (SPE) and ultrafiltration were simultaneously evaluated using non-spiked and metabolite standards spiked human plasma. Sample-to-extraction solvent ratio and metabolites derivatization conditions were also investigated and optimised. The results demonstrated that stepwise LLE protocol (MeOH-H2O/CHCl3/CHCl3-MeOH) was the most efficient extraction strategy for the combination of untargeted and targeted metabolomics. Sample-to-extraction solvent ratio of 1:3 (v:v) was found to perform better than higher solvent ratios. Also, the maximum derivatization performance was obtained when using 30 µL N-methyl-N-tri-methylsilyltrifluoroacetamide (MSTFA) and 1% trimethyl-chlorosilane (TMCS) incubated at 60 °C for 120 min. In the end, the optimised sample preparation method was applied successfully to plasma untargeted metabolomics of rheumatoid arthritis (RA) and four metabolites (gamma-butyrolactone, MG (0:0/18:0/0:0), tocopherol and oxalic acid) were found to be potential biomarkers for the diagnosis of RA.
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Artritis Reumatoide , Metabolómica , Artritis Reumatoide/diagnóstico , Cromatografía de Gases y Espectrometría de Masas , Humanos , Plasma , Flujo de TrabajoRESUMEN
Liquid chromatography tandem mass spectrometry has been a widely used technique for quantifying oligonucleotides in biological samples. However, lack of simple and efficient sample cleanup approach remains a challenge. Our study aimed to evaluate the major factors during the sample pretreatment process for developing optimal sample preparation workflow for oligonucleotides. In this study, we have employed a model formed with rat plasma containing a 16 mer oligonucleotide standard in order to comprehensively optimize the sample preparation procedures. These included liquid-liquid extraction (LLE), solid-phase extraction (SPE), protein precipitation (PPT) and LLE combined with SPE. LLE with phenol: dichloromethane (2:1, v:v) was found to be the most efficient sample cleanup procedure with low cost and less toxicity. Followed by the extraction, ethanol precipitation (-80 °C, 5 min) was determined to be the optimal drying conditions. Also, mass spectrometric parameters were tuned to optimal conditions. It was found that the central composite design suite was proved to be highly practical for optimizing MS parameters. Finally, the thoroughly optimized sample preparation workflow was fully validated. The developed assay provided a quantitative range of 0.25-1000 nM, with accuracy and precision were < 7.45% and < 12.20%, respectively. Matrix effect and carryover were also evaluated and no significant effect was observed.
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Métodos Analíticos de la Preparación de la Muestra , Oligonucleótidos/análisis , Espectrometría de Masas en Tándem/métodos , Adsorción , Animales , Cloroformo/química , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida/métodos , Desecación , Glucógeno/química , Concentración de Iones de Hidrógeno , Extracción Líquido-Líquido , Cloruro de Metileno/química , Fenol/química , Fosfolípidos/aislamiento & purificación , Ratas , Reproducibilidad de los Resultados , Extracción en Fase Sólida , Soluciones , Flujo de TrabajoRESUMEN
Lack of clinically specific biomarkers has impeded the diagnosis of osteoarthritis (OA) and limited understanding of pathogenesis for OA has also restrained the enhancement of therapeutic measures. In the study, plasma untargeted metabolomics of twelve OA patients and twenty healthy controls (HC) were analyzed by gas chromatography coupled with quadrupole time-of-flight mass spectrometry (GC/Q-TOF-MS). The differential metabolites (DMs) between OA and HC were evaluated by multivariate analysis and Bayes discriminant analysis was employed to discover potential diagnosis biomarkers. Meanwhile a transcriptomic dataset GSE55235 was downloaded from GEO database to explore the differentially expressed genes (DEGs) between OA and HC by R/Bioconductor project. Finally, an integrative analysis of DMs and DEGs was performed to investigate the possible molecular mechanisms of OA. As a result, a panel of three metabolites including succinic acid, xanthurenic acid and L-tryptophan was revealed to potentially act as biomarker for the diagnosis of OA. Furthermore, the integrated analysis of metabolomics and transcriptomics showed the top three enrichment in the T cell receptor signaling pathway, Fc epsilon RI (FcεRI) signaling pathway, and thermogenesis, explaining the inflammation, joint destruction and energy metabolism disorders in OA.
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Osteoartritis , Transcriptoma , Teorema de Bayes , Biomarcadores , Humanos , Metabolómica , Osteoartritis/diagnóstico , Osteoartritis/genéticaRESUMEN
AIMS: To investigate the effect of ethanol intake on the whole enterohepatic circulation (EHC) of bile acids (BAs) and, more importantly, on pharmacokinetics of irinotecan. METHODS: The present study utilized a mouse model administered by gavage with 0 (control), 240 mg/100 g (30%, v/v) and 390 mg/100 g (50%, v/v) ethanol for 6 weeks, followed by BA profiles in the whole EHC (including liver, gallbladder, intestine and plasma) and colon using ultra-high performance liquid chromatography with tandem mass spectrometry analysis. Pharmacokinetic parameters of irinotecan were measured after administration of irinotecan (i.v. 5 mg/kg) on alcohol-treated mice. RESULTS: The results showed that compared with the control group, concentrations of most free-BAs, total amount of the three main forms of BAs (free-BA, taurine-BA and glycine-BA) and total BAs (TBAs) in 50% ethanol intake group were significantly increased, which are mostly attributed to the augmentation of free-BAs and taurine-BAs. Additionally, the TBAs in liver and gallbladder and the BA pool were markedly increased in the 30% ethanol intake group. Importantly, ethanol intake upregulated the expression of BA-related enzymes (Cyp7a1, Cyp27a1, Cyp8b1 and Baat) and transporters (Bsep, Mrp2, P-gp and Asbt) and downregulated the expression of transporter Ntcp and nuclear receptor Fxr in the liver and ileum, respectively. Additionally, 50% ethanol intake caused fairly distinct liver injury. Furthermore, the AUC0-24 h of irinotecan and SN38 were significantly reduced but their clearance was significantly increased in the disrupted EHC of BA by 50% ethanol intake. CONCLUSIONS: The present study demonstrated that ethanol intake altered the expression of BA-related synthetases and transporters. The BA levels, especially the toxic BAs (chenodeoxycholic acid, deoxycholic acid and lithocholic acid), in the whole EHC were significantly increased by ethanol intake, which may provide a potential explanation to illuminate the pathogenesis of alcoholic liver injury. Most importantly, chronic ethanol consumption had a significant impact on the pharmacokinetics (AUC0-24 h and clearance) of irinotecan and SN38; hence colon cancer patients with chronic alcohol consumption treated with irinotecan deserve our close attention.
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Antineoplásicos/farmacocinética , Ácidos y Sales Biliares/metabolismo , Circulación Enterohepática/efectos de los fármacos , Irinotecán/farmacocinética , Consumo de Bebidas Alcohólicas , Animales , Ácidos y Sales Biliares/sangre , Western Blotting , Colon/efectos de los fármacos , Colon/patología , Modelos Animales de Enfermedad , Vesícula Biliar/efectos de los fármacos , Vesícula Biliar/patología , Irinotecán/farmacología , Hígado/efectos de los fármacos , Hígado/patología , Masculino , RatonesRESUMEN
LC-MS-based untargeted metabolomics have been proven to be an extremely promising technique to discover biomarkers and explore the mechanisms underlying diseases, which, however, relies heavily on sample pretreatment for metabolite extraction. In the present study, a systematic and pragmatic evaluation of eight protocols employing conventional metabolites extraction strategies, protein precipitation (PPT), and liquid-liquid extraction (LLE), with and without proteinase K (PK) incubation, was performed simultaneously, using human plasma and a mixture of 39 endogenous metabolite standards. These protocols were as follows: (1) PPT with methanol, (2) PPT with acetonitrile, (3) PPT with 2-propanol, (4) two-step LLE of CH2Cl2-MeOH, followed by MeOH-H2O, (5) PK incubation combining two-step LLE of CH2Cl2-MeOH followed by MeOH-H2O, (6) two-step LLE of CHCl3-MeOH, followed by MeOH-H2O, (7) PK incubation combining two-step LLE of CHCl3-MeOH, followed by MeOH-H2O, (8) PK incubation combining MeOH-EtOH PPT. The results suggested that two-step LLE produced broader metabolome coverage than protein precipitation, and the addition of proteinase K enhanced the extraction performance further. Taken together, PK incubation combining two-step LLE of CHCl3-MeOH, followed by MeOH-H2O, was determined to be the most suitable extraction method, because of its broad metabolome coverage, high reproducibility, and satisfactory recovery. Next, the developed optimal sample preparation method was applied successfully to profile the plasma metabolome of colorectal adenoma and uncover its potential mechanism for significant differential changes in linoleic acid and phospholipid metabolism.
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Adenoma/metabolismo , Endopeptidasa K/química , Enfermedades Gastrointestinales/metabolismo , Metaboloma , Plasma/metabolismo , Adulto , Análisis Químico de la Sangre/métodos , Cromatografía Líquida de Alta Presión/métodos , Femenino , Humanos , Extracción Líquido-Líquido/métodos , Masculino , Metabolómica/métodos , Persona de Mediana Edad , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
The effect of sample containers made of different materials on the MS-based analysis of oligonucleotides remains unknown. Here, we evaluated five types of sample containers on the MS signal stability of oligonucleotide, and they were normal glass insert, silanized glass insert with three different silanization techniques, and polypropylene sample vial. Also, we attempted to tackle signal stability issue by varying modifiers in dissolution solvent. Our results showed that sample containers made of different materials can significantly influence the MS signal stability of oligonucleotide at low concentration. Triethylamine (TEA) evidently improved both the signal stability and intensity of oligonucleotide.
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Etilaminas/química , Oligonucleótidos/análisis , Espectrometría de MasasRESUMEN
RATIONALE: Hexafluoroisopropanol (HFIP) has been widely used as a counter anion in the mobile phase for ion-pairing reversed-phase liquid chromatography/mass spectrometry (IP-RP-LC/MS) analysis of oligonucleotides. However, researchers are still searching for improvements to counter anions for LC/MS analysis of oligonucleotides. This study aimed to find alternatives to HFIP for analyzing oligonucleotides. METHODS: The study was performed using an Agilent 1290 ultra-high-performance liquid chromatography (UHPLC) system coupled to an Agilent 6540 mass spectrometer by using an oligonucleotide BEH C18 column (100 × 2.1 mm, 1.7 µm). Buffer systems containing ion-pairing reagents (triethylamine, tripropylamine, hexylamine, dimethylbutylamine, diisopropylethylamine, N,N-dimethylcyclohexylamine, and octylamine) and fluoroalcohols (HFIP and hexafluoro-2-methyl-2-propanol (HFTP)) were compared chromatographically and mass spectrometrically. RESULTS: Results showed that HFTP has better desalting ability than HFIP, but both HFIP and HFTP have comparable effects on the separation of oligonucleotides sized from 10mer to 40mer for most of ion-pairing reagents, with the exception of triethylamine and N,N-dimethylcyclohexylamine, where HFIP performed better than HFTP. CONCLUSIONS: The choice of fluoroalcohols in IP-RP-LC/MS analysis of oligonucleotides depends on the type of ion-pairing reagents used in the mobile phase. As a guideline, we would recommend to use either HA-HFIP or HA-HFTP for small oligonucleotides, but TPA-HFTP for large oligonucleotides for IP-RP-LC/MS analysis of synthetic oligonucleotides.
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Cromatografía de Fase Inversa/métodos , Oligonucleótidos/análisis , Propanoles/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Aniones/químicaRESUMEN
RATIONALE: Hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry (HILIC-LC/ESI-MS) has been proved to be useful for the quality control of oligonucleotides. However, the lack of separation for some oligonucleotides using HILIC-LC/MS has proved problematic. This study aimed to improve the resolving ability of HILIC-LC/MS. METHODS: The study was performed on a Waters UPLC® system coupled to a Waters LCT premier XE ESI-TOF mass spectrometer using a Zorbax® RRHD HILIC column (2.1 mm × 100 mm, 1.8 µm). Buffer systems contained triethylammonium acetate (TEAA) and acetonitrile. The effects of the concentration of TEAA and the type of organic modifiers on the separation of oligonucleotides were investigated. RESULTS: The results showed that the optimum concentration of TEAA is 10 mM and acetonitrile is a better organic solvent than methanol. The addition of TEAA in the HILIC mobile phase improved the separation of N from N + A significantly compared to the HILIC method buffered with ammonium acetate. The IP-HILIC chromatography has demonstrated that the separation of oligonucleotides is sequence dependent. In addition, the IP-HILIC method produces a much simpler mass spectrum of an oligonucleotide with very efficient desalting. CONCLUSIONS: The HILIC-LC/MS method with the addition of TEAA at a MS-compatible concentration has improved the separation of oligonucleotides. The IP-HILIC-LC/MS method also produces very simple mass spectra with high desalting efficiency.