Editorial

3 pages, 196 KiB

Open AccessEditorial

Advances in Metabolic Profiling of Biological Samples

by Joana Pinto

Metabolites 2023, 13(4), 534; https://doi.org/10.3390/metabo13040534 - 09 Apr 2023

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Metabolomics constitutes a promising approach to clinical diagnostics, but its practical implementation in clinical settings is hindered by the requirement for rapid and efficient analytical methods [...] Full article

(This article belongs to the Special Issue Advances in Metabolic Profiling of Biological Samples)

Research

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15 pages, 4181 KiB

Open AccessArticle

The Role of the Preanalytical Step for Human Saliva Analysis via Vibrational Spectroscopy

by Beatrice Campanella, Stefano Legnaioli, Massimo Onor, Edoardo Benedetti and Emilia Bramanti

Metabolites 2023, 13(3), 393; https://doi.org/10.3390/metabo13030393 - 08 Mar 2023

Cited by 1 | Viewed by 1419

Abstract

Saliva is an easily sampled matrix containing a variety of biochemical information, which can be correlated with the individual health status. The fast, straightforward analysis of saliva by vibrational (ATR-FTIR and Raman) spectroscopy is a good premise for large-scale preclinical studies to aid [...] Read more.

Saliva is an easily sampled matrix containing a variety of biochemical information, which can be correlated with the individual health status. The fast, straightforward analysis of saliva by vibrational (ATR-FTIR and Raman) spectroscopy is a good premise for large-scale preclinical studies to aid translation into clinics. In this work, the effects of saliva collection (spitting/swab) and processing (two different deproteinization procedures) were explored by principal component analysis (PCA) of ATR-FTIR and Raman data and by investigating the effects on the main saliva metabolites by reversed-phase chromatography (RPC-HPLC-DAD). Our results show that, depending on the bioanalytical information needed, special care must be taken when saliva is collected with swabs because the polymeric material significantly interacts with some saliva components. Moreover, the analysis of saliva before and after deproteinization by FTIR and Raman spectroscopy allows to obtain complementary biological information. Full article

(This article belongs to the Special Issue Advances in Metabolic Profiling of Biological Samples)

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15 pages, 2719 KiB

Open AccessArticle

HILIC-MS for Untargeted Profiling of the Free Glycation Product Diversity

by Yingfei Yan, Daniel Hemmler and Philippe Schmitt-Kopplin

Metabolites 2022, 12(12), 1179; https://doi.org/10.3390/metabo12121179 - 25 Nov 2022

Cited by 4 | Viewed by 1792

Abstract

Glycation products produced by the non-enzymatic reaction between reducing carbohydrates and amino compounds have received increasing attention in both food- and health-related research. Although liquid chromatography mass spectrometry (LC-MS) methods for analyzing glycation products already exist, only a few common advanced glycation end [...] Read more.

Glycation products produced by the non-enzymatic reaction between reducing carbohydrates and amino compounds have received increasing attention in both food- and health-related research. Although liquid chromatography mass spectrometry (LC-MS) methods for analyzing glycation products already exist, only a few common advanced glycation end products (AGEs) are usually covered by quantitative methods. Untargeted methods for comprehensively analyzing glycation products are still lacking. The aim of this study was to establish a method for simultaneously characterizing a wide range of free glycation products using the untargeted metabolomics approach. In this study, Maillard model systems consisting of a multitude of heterogeneous free glycation products were chosen for systematic method optimization, rather than using a limited number of standard compounds. Three types of hydrophilic interaction liquid chromatography (HILIC) columns (zwitterionic, bare silica, and amide) were tested due to their good retention for polar compounds. The zwitterionic columns showed better performance than the other two types of columns in terms of the detected feature numbers and detected free glycation products. Two zwitterionic columns were selected for further mobile phase optimization. For both columns, the neutral mobile phase provided better peak separation, whereas the acidic condition provided a higher quality of chromatographic peak shapes. The ZIC-cHILIC column operating under acidic conditions offered the best potential to discover glycation products in terms of providing good peak shapes and maintaining comparable compound coverage. Finally, the optimized HILIC-MS method can detect 70% of free glycation product features despite interference from the complex endogenous metabolites from biological matrices, which showed great application potential for glycation research and can help discover new biologically important glycation products. Full article

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14 pages, 1150 KiB

Open AccessEditor’s ChoiceArticle

Integration of Liver Glycogen and Triglyceride NMR Isotopomer Analyses Provides a Comprehensive Coverage of Hepatic Glucose and Fructose Metabolism

by Ivan Viegas, Giada Di Nunzio, Getachew D. Belew, Alejandra N. Torres, João G. Silva, Luis Perpétuo, Cristina Barosa, Ludgero C. Tavares and John G. Jones

Metabolites 2022, 12(11), 1142; https://doi.org/10.3390/metabo12111142 - 19 Nov 2022

Cited by 2 | Viewed by 3738

Abstract

Dietary glucose and fructose are both efficiently assimilated by the liver but a comprehensive measurement of this process starting from their conversion to sugar phosphates, involvement of the pentose phosphate pathway (PPP), and conversion to glycogen and lipid storage products, remains incomplete. Mice [...] Read more.

Dietary glucose and fructose are both efficiently assimilated by the liver but a comprehensive measurement of this process starting from their conversion to sugar phosphates, involvement of the pentose phosphate pathway (PPP), and conversion to glycogen and lipid storage products, remains incomplete. Mice were fed a chow diet supplemented with 35 g/100 mL drinking water of a 55/45 fructose/glucose mixture for 18 weeks. On the final night, the sugar mixture was enriched with either [U-¹³C]glucose or [U-¹³C]fructose, and deuterated water (²H₂O) was also administered. ¹³C-isotopomers representing newly synthesized hepatic glucose-6-phosphate (glucose-6-P), glycerol-3-phosphate, and lipogenic acetyl-CoA were quantified by ²H and ¹³C NMR analysis of post-mortem liver glycogen and triglyceride. These data were applied to a metabolic model covering glucose-6-P, PPP, triose-P, and de novo lipogenesis (DNL) fluxes. The glucose supplement was converted to glucose-6-P via the direct pathway, while the fructose supplement was metabolized by the liver to gluconeogenic triose-P via fructokinase–aldolase–triokinase. Glucose-6-P from all carbohydrate sources accounted for 40–60% of lipogenic acetyl-CoA and 10–12% was oxidized by the pentose phosphate pathway (PPP). The yield of NADPH from PPP flux accounted for a minority (~30%) of the total DNL requirement. In conclusion, this approach integrates measurements of glucose-6-P, PPP, and DNL fluxes to provide a holistic and informative assessment of hepatic glucose and fructose metabolism. Full article

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10 pages, 3060 KiB

Open AccessArticle

Lipid Serum Profiling of Boar-Tainted and Untainted Pigs Using GC×GC–TOFMS: An Exploratory Study

by Kinjal Bhatt, Thibaut Dejong, Lena M. Dubois, Alice Markey, Nicolas Gengler, José Wavreille, Pierre-Hugues Stefanuto and Jean-François Focant

Metabolites 2022, 12(11), 1111; https://doi.org/10.3390/metabo12111111 - 15 Nov 2022

Cited by 3 | Viewed by 1786

Abstract

Mass spectrometry (MS)-based techniques, including liquid chromatography coupling, shotgun lipidomics, MS imaging, and ion mobility, are widely used to analyze lipids. However, with enhanced separation capacity and an optimized chemical derivatization approach, comprehensive two-dimensional gas chromatography (GC×GC) can be a powerful tool to [...] Read more.

Mass spectrometry (MS)-based techniques, including liquid chromatography coupling, shotgun lipidomics, MS imaging, and ion mobility, are widely used to analyze lipids. However, with enhanced separation capacity and an optimized chemical derivatization approach, comprehensive two-dimensional gas chromatography (GC×GC) can be a powerful tool to investigate some groups of small lipids in the framework of lipidomics. This study describes the optimization of a dedicated two-stage derivatization and extraction process to analyze different saturated and unsaturated fatty acids in plasma by two-dimensional gas chromatography–time-of-flight mass spectrometry (GC×GC–TOFMS) using a full factorial design. The optimized condition has a composite desirability of 0.9159. This optimized sample preparation and chromatographic condition were implemented to differentiate between positive (BT) and negative (UT) boar-tainted pigs based on fatty acid profiling in pig serum using GC×GC–TOFMS. A chemometric screening, including unsupervised (PCA, HCA) and supervised analysis (PLS–DA), as well as univariate analysis (volcano plot), was performed. The results suggested that the concentration of PUFA ω-6 and cholesterol derivatives were significantly increased in BT pigs, whereas SFA and PUFA ω-3 concentrations were increased in UT pigs. The metabolic pathway and quantitative enrichment analysis suggest the significant involvement of linolenic acid metabolism. Full article

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13 pages, 1609 KiB

Open AccessArticle

Mapping of Urinary Volatile Organic Compounds by a Rapid Analytical Method Using Gas Chromatography Coupled to Ion Mobility Spectrometry (GC–IMS)

by Giulia Riccio, Silvia Baroni, Andrea Urbani and Viviana Greco

Metabolites 2022, 12(11), 1072; https://doi.org/10.3390/metabo12111072 - 05 Nov 2022

Cited by 2 | Viewed by 1620

Abstract

Volatile organic compounds (VOCs) are a differentiated class of molecules, continuously generated in the human body and released as products of metabolic pathways. Their concentrations vary depending on pathophysiological conditions. They are detectable in a wide variety of biological samples, such as exhaled [...] Read more.

Volatile organic compounds (VOCs) are a differentiated class of molecules, continuously generated in the human body and released as products of metabolic pathways. Their concentrations vary depending on pathophysiological conditions. They are detectable in a wide variety of biological samples, such as exhaled breath, faeces, and urine. In particular, urine represents an easily accessible specimen widely used in clinics. The most used techniques for VOCs detections are expensive and time-consuming, thus not allowing for rapid clinical analysis. In this perspective, the aim of this study is a comprehensive characterisation of the urine volatilome by the development of an alternative rapid analytical method. Briefly, 115 urine samples are collected; sample treatment is not needed. VOCs are detected in the urine headspace using gas chromatography coupled to ion mobility spectrometry (GC–IMS) by an extremely fast analysis (10 min). The method is analytically validated; the analysis is sensitive and robust with results comparable to those reported with other techniques. Twenty-three molecules are identified, including ketones, aldehydes, alcohols, and sulphur compounds, whose concentration is altered in several pathological states such as cancer and metabolic disorders. Therefore, it opens new perspectives for fast diagnosis and screening, showing great potential for clinical applications. Full article

(This article belongs to the Special Issue Advances in Metabolic Profiling of Biological Samples)

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17 pages, 2720 KiB

Open AccessEditor’s ChoiceArticle

Combining Feature-Based Molecular Networking and Contextual Mass Spectral Libraries to Decipher Nutrimetabolomics Profiles

by Lapo Renai, Marynka Ulaszewska, Fulvio Mattivi, Riccardo Bartoletti, Massimo Del Bubba and Justin J. J. van der Hooft

Metabolites 2022, 12(10), 1005; https://doi.org/10.3390/metabo12101005 - 21 Oct 2022

Cited by 4 | Viewed by 2302

Abstract

Untargeted metabolomics approaches deal with complex data hindering structural information for the comprehensive analysis of unknown metabolite features. We investigated the metabolite discovery capacity and the possible extension of the annotation coverage of the Feature-Based Molecular Networking (FBMN) approach by adding two novel [...] Read more.

Untargeted metabolomics approaches deal with complex data hindering structural information for the comprehensive analysis of unknown metabolite features. We investigated the metabolite discovery capacity and the possible extension of the annotation coverage of the Feature-Based Molecular Networking (FBMN) approach by adding two novel nutritionally-relevant (contextual) mass spectral libraries to the existing public ones, as compared to widely-used open-source annotation protocols. Two contextual mass spectral libraries in positive and negative ionization mode of ~300 reference molecules relevant for plant-based nutrikinetic studies were created and made publicly available through the GNPS platform. The postprandial urinary metabolome analysis within the intervention of Vaccinium supplements was selected as a case study. Following the FBMN approach in combination with the added contextual mass spectral libraries, 67 berry-related and human endogenous metabolites were annotated, achieving a structural annotation coverage comparable to or higher than existing non-commercial annotation workflows. To further exploit the quantitative data obtained within the FBMN environment, the postprandial behavior of the annotated metabolites was analyzed with Pearson product-moment correlation. This simple chemometric tool linked several molecular families with phase II and phase I metabolism. The proposed approach is a powerful strategy to employ in longitudinal studies since it reduces the unknown chemical space by boosting the annotation power to characterize biochemically relevant metabolites in human biofluids. Full article

(This article belongs to the Special Issue Advances in Metabolic Profiling of Biological Samples)

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16 pages, 1481 KiB

Open AccessEditor’s ChoiceArticle

A Sample Preparation Method for the Simultaneous Profiling of Signaling Lipids and Polar Metabolites in Small Quantities of Muscle Tissues from a Mouse Model for Sarcopenia

by Yupeng He, Marlien van Mever, Wei Yang, Luojiao Huang, Rawi Ramautar, Yvonne Rijksen, Wilbert P. Vermeij, Jan H. J. Hoeijmakers, Amy C. Harms, Peter W. Lindenburg and Thomas Hankemeier

Metabolites 2022, 12(8), 742; https://doi.org/10.3390/metabo12080742 - 12 Aug 2022

Cited by 1 | Viewed by 2178

Abstract

The metabolic profiling of a wide range of chemical classes relevant to understanding sarcopenia under conditions in which sample availability is limited, e.g., from mouse models, small muscles, or muscle biopsies, is desired. Several existing metabolomics platforms that include diverse classes of signaling [...] Read more.

The metabolic profiling of a wide range of chemical classes relevant to understanding sarcopenia under conditions in which sample availability is limited, e.g., from mouse models, small muscles, or muscle biopsies, is desired. Several existing metabolomics platforms that include diverse classes of signaling lipids, energy metabolites, and amino acids and amines would be informative for suspected biochemical pathways involved in sarcopenia. The sample limitation requires an optimized sample preparation method with minimal losses during isolation and handling and maximal accuracy and reproducibility. Here, two developed sample preparation methods, BuOH-MTBE-Water (BMW) and BuOH-MTBE-More-Water (BMMW), were evaluated and compared with previously reported methods, Bligh-Dyer (BD) and BuOH-MTBE-Citrate (BMC), for their suitability for these classes. The most optimal extraction was found to be the BMMW method, with the highest extraction recovery of 63% for the signaling lipids and 81% for polar metabolites, and an acceptable matrix effect (close to 1.0) for all metabolites of interest. The BMMW method was applied on muscle tissues as small as 5 mg (dry weight) from the well-characterized, prematurely aging, DNA repair-deficient Ercc1^∆/− mouse mutant exhibiting multiple–morbidities, including sarcopenia. We successfully detected 109 lipids and 62 polar targeted metabolites. We further investigated whether fast muscle tissue isolation is necessary for mouse sarcopenia studies. A muscle isolation procedure involving 15 min at room temperature revealed a subset of metabolites to be unstable; hence, fast sample isolation is critical, especially for more oxidative muscles. Therefore, BMMW and fast muscle tissue isolation are recommended for future sarcopenia studies. This research provides a sensitive sample preparation method for the simultaneous extraction of non-polar and polar metabolites from limited amounts of muscle tissue, supplies a stable mouse muscle tissue collection method, and methodologically supports future metabolomic mechanistic studies of sarcopenia. Full article

(This article belongs to the Special Issue Advances in Metabolic Profiling of Biological Samples)

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17 pages, 1475 KiB

Open AccessArticle

Simultaneous Quantification of Steroid Hormones Using hrLC-MS in Endocrine Tissues of Male Rats and Human Samples

by Guillermo Bordanaba-Florit, Sebastiaan van Liempd, Diana Cabrera, Félix Royo and Juan Manuel Falcón-Pérez

Metabolites 2022, 12(8), 714; https://doi.org/10.3390/metabo12080714 - 30 Jul 2022

Cited by 6 | Viewed by 1834

Abstract

Steroid hormones play a vital role in the regulation of cellular processes, and dysregulation of these metabolites can provoke or aggravate pathological issues, such as autoimmune diseases and cancer. Regulation of steroid hormones involves different organs and biological compartments. Therefore, it is important [...] Read more.

Steroid hormones play a vital role in the regulation of cellular processes, and dysregulation of these metabolites can provoke or aggravate pathological issues, such as autoimmune diseases and cancer. Regulation of steroid hormones involves different organs and biological compartments. Therefore, it is important to accurately determine their levels in tissues and biofluids to monitor changes after challenge or during disease. In this work, we have developed and optimized the extraction and quantification of 11 key members of the different steroid classes, including androgens, estrogens, progestogens and corticoids. The assay consists of a liquid/liquid extraction step and subsequent quantification by high-resolution liquid chromatography coupled time-of-flight mass spectrometry. The recoveries range between 74.2 to 126.9% and 54.9 to 110.7%, using a cell culture or urine as matrix, respectively. In general, the signal intensity loss due to matrix effect is no more than 30%. The method has been tested in relevant steroidogenic tissues in rat models and it has also been tested in human urine samples. Overall, this assay measures 11 analytes simultaneously in 6 min runtime and it has been applied in adrenal gland, testis, prostate, brain and serum from rats, and urine and extracellular vesicles from humans. Full article

(This article belongs to the Special Issue Advances in Metabolic Profiling of Biological Samples)

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15 pages, 2635 KiB

Open AccessArticle

An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells

by Camillo Morano, Aida Zulueta, Anna Caretti, Gabriella Roda, Rita Paroni and Michele Dei Cas

Metabolites 2022, 12(5), 450; https://doi.org/10.3390/metabo12050450 - 17 May 2022

Cited by 9 | Viewed by 1845

Abstract

The main concerns in targeted “sphingolipidomics” are the extraction and proper handling of biological samples to avoid interferences and achieve a quantitative yield well representing all the sphingolipids in the matrix. Our work aimed to compare different pre-analytical procedures and to [...] Read more.

The main concerns in targeted “sphingolipidomics” are the extraction and proper handling of biological samples to avoid interferences and achieve a quantitative yield well representing all the sphingolipids in the matrix. Our work aimed to compare different pre-analytical procedures and to evaluate a derivatization step for sphingoid bases quantification, to avoid interferences and improve sensitivity. We tested four protocols for the extraction of sphingolipids from human plasma, at different temperatures and durations, and two derivatization procedures for the conversion of sphingoid bases into phenylthiourea derivatives. Different columns and LC-MS/MS chromatographic conditions were also tested. The protocol that worked better for sphingolipids analysis involved a single-phase extraction in methanol/chloroform mixture (2:1, v/v) for 1 h at 38 °C, followed by a 2 h alkaline methanolysis at 38 °C, for the suppression of phospholipids signals. The derivatization of sphingoid bases promotes the sensibility of non-phosphorylated species but we proved that it is not superior to a careful choice of the appropriate column and a full-length elution gradient. Our procedure was eventually validated by analyzing plasma and erythrocyte samples of 20 volunteers. While both extraction and methanolysis are pivotal steps, our final consideration is to analyze sphingolipids and sphingoid bases under different chromatographic conditions, minding the interferences. Full article

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15 pages, 4368 KiB

Open AccessArticle

The Human Meconium Metabolome and Its Evolution during the First Days of Life

by Nihel Bekhti, Florence Castelli, Alain Paris, Blanche Guillon, Christophe Junot, Clémence Moiron, François Fenaille and Karine Adel-Patient

Metabolites 2022, 12(5), 414; https://doi.org/10.3390/metabo12050414 - 05 May 2022

Cited by 3 | Viewed by 1868

Abstract

Meconium represents the first newborn stools, formed from the second month of gestation and excreted in the first days after birth. As an accumulative and inert matrix, it accumulates most of the molecules transferred through the placenta from the mother to the fetus [...] Read more.

Meconium represents the first newborn stools, formed from the second month of gestation and excreted in the first days after birth. As an accumulative and inert matrix, it accumulates most of the molecules transferred through the placenta from the mother to the fetus during the last 6 months of pregnancy, and those resulting from the metabolic activities of the fetus. To date, only few studies dealing with meconium metabolomics have been published. In this study, we aimed to provide a comprehensive view of the meconium metabolic composition using 33 samples collected longitudinally from 11 healthy newborns and to analyze its evolution during the first 3 days of life. First, a robust and efficient methodology for metabolite extraction was implemented. Data acquisition was performed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), using two complementary LC-HRMS conditions. Data preprocessing and treatment were performed using the Workflow4Metabolomics platform and the metabolite annotation was performed using our in-house database by matching accurate masses, retention times, and MS/MS spectra to those of pure standards. We successfully identified up to 229 metabolites at a high confidence level in human meconium, belonging to diverse chemical classes and from different origins. A progressive evolution of the metabolic profile was statistically evidenced, with sugars, amino acids, and some bacteria-derived metabolites being among the most impacted identified compounds. Our implemented analytical workflow allows a unique and comprehensive description of the meconium metabolome, which is related to factors, such as maternal diet and environment. Full article

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15 pages, 2050 KiB

Open AccessProtocol

Quantitative Profiling of Bile Acids in Feces of Humans and Rodents by Ultra-High-Performance Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

by Xiaoxu Zhang, Xiaoxue Liu, Jiufang Yang, Fazheng Ren and Yixuan Li

Metabolites 2022, 12(7), 633; https://doi.org/10.3390/metabo12070633 - 11 Jul 2022

Cited by 5 | Viewed by 1989

Abstract

A simple, sensitive, and reliable quantification and identification method was developed and validated for simultaneous analysis of 58 bile acids (BAs) in human and rodent (mouse and rat) fecal samples. The method involves an extraction step with a 5% ammonium–ethanol aqueous solution; the [...] Read more.

A simple, sensitive, and reliable quantification and identification method was developed and validated for simultaneous analysis of 58 bile acids (BAs) in human and rodent (mouse and rat) fecal samples. The method involves an extraction step with a 5% ammonium–ethanol aqueous solution; the BAs were quantified by high-resolution mass spectrometry (ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry, UPLC–Q-TOF). The recoveries were 80.05–120.83%, with coefficient variations (CVs) of 0.01–9.82% for three biological species. The limits of detection (LODs) were in the range of 0.01–0.24 μg/kg, and the limits of quantification (LOQs) ranged from 0.03 to 0.81 μg/kg. In addition, the analytical method was used to identify and quantify BAs in end-stage renal disease (ESRD) patients, C57BL/6 mice, and Sprague-Dawley (SD) rats. The fecal BA profile and analysis of BA indices in these samples provide valuable information for further BA metabolic disorder research. Full article

(This article belongs to the Special Issue Advances in Metabolic Profiling of Biological Samples)

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