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

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 Delta/-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.Analytical BioScience

PRMT3 inhibitor SGC707 reduces triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice

Protein arginine methyltransferase 3 (PRMT3) is a co-activator of liver X receptor capable of selectively modulating hepatic triglyceride synthesis. Here we investigated whether pharmacological PRMT3 inhibition can diminish the hepatic steatosis extent and lower plasma lipid levels and atherosclerosis susceptibility. Hereto, male hyperlipidemic low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet and injected 3 times per week intraperitoneally with PRMT3 inhibitor SGC707 or solvent control. Three weeks into the study, SGC707-treated mice developed severe pruritus and scratching-associated skin lesions, leading to early study termination. SGC707-treated mice exhibited 50% lower liver triglyceride stores as well as 32% lower plasma triglyceride levels. Atherosclerotic lesions were virtually absent in all experimental mice. Plasma metabolite analysis revealed that levels of taurine-conjugated bile acids were ~ threefold increased (P < 0.001) in response to SGC707 treatment, which was paralleled by systemically higher bile acid receptor TGR5 signalling. In conclusion, we have shown that SGC707 treatment reduces hepatic steatosis and plasma triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice. These findings suggest that pharmacological PRMT3 inhibition can serve as therapeutic approach to treat non-alcoholic fatty liver disease and dyslipidemia/atherosclerosis, when unwanted effects on cholesterol and bile acid metabolism can be effectively tackled

PRMT3 inhibitor SGC707 reduces triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice

Protein arginine methyltransferase 3 (PRMT3) is a co-activator of liver X receptor capable of selectively modulating hepatic triglyceride synthesis. Here we investigated whether pharmacological PRMT3 inhibition can diminish the hepatic steatosis extent and lower plasma lipid levels and atherosclerosis susceptibility. Hereto, male hyperlipidemic low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet and injected 3 times per week intraperitoneally with PRMT3 inhibitor SGC707 or solvent control. Three weeks into the study, SGC707-treated mice developed severe pruritus and scratching-associated skin lesions, leading to early study termination. SGC707-treated mice exhibited 50% lower liver triglyceride stores as well as 32% lower plasma triglyceride levels. Atherosclerotic lesions were virtually absent in all experimental mice. Plasma metabolite analysis revealed that levels of taurine-conjugated bile acids were ~ threefold increased (P < 0.001) in response to SGC707 treatment, which was paralleled by systemically higher bile acid receptor TGR5 signalling. In conclusion, we have shown that SGC707 treatment reduces hepatic steatosis and plasma triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice. These findings suggest that pharmacological PRMT3 inhibition can serve as therapeutic approach to treat non-alcoholic fatty liver disease and dyslipidemia/atherosclerosis, when unwanted effects on cholesterol and bile acid metabolism can be effectively tackled.Analytical BioScience

Three-Phase Electroextraction: A New (Online) Sample Purification and Enrichment Method for Bioanalysis

The migration and at the same time enrichment of analytes from a liquid aqueous sample donor phase through an immiscible organic solvent layer acting as a filter phase into a liquid aqueous acceptor phase is enabled by the application of an electric field between the donor and acceptor phase. The organic filter phase acts as a purification filter, which prevents, for example, proteins from migrating into the acceptor phase. Moreover, the composition of the organic filter phase influences the selectivity of the extraction. We show that analytes can be rapidly enriched from a 50 μL donor phase at the bottom of a sample vial, via an immiscible organic filter phase, into a 2 μL acceptor phase which consists of a droplet that is hanging from a (conductive) pipet tip in the organic filter phase. Acylcarnitines spiked to human plasma as a donor phase were extracted reproducibly with good linearity and a 10-fold improved limit of detection and, importantly, resulted in a stable, protein-free nanoelectrospray signal. Finally, a proof of principle toward the online integration in an automated nanoelectrospray-direct infusion-mass spectrometry platform has been realized. This makes 3-phase electroextraction (3-phase EE) a novel sample purification and enrichment method, with straightforward online integration possibility. We envision that 3-phase EE will enable new possibilities using electrokinetic sample pretreatment for fully automated, high-throughput bioanalysis purposes

Three-Phase Electroextraction: A New (Online) Sample Purification and Enrichment Method for Bioanalysis

The migration and at the same time enrichment of analytes from a liquid aqueous sample donor phase through an immiscible organic solvent layer acting as a filter phase into a liquid aqueous acceptor phase is enabled by the application of an electric field between the donor and acceptor phase. The organic filter phase acts as a purification filter, which prevents, for example, proteins from migrating into the acceptor phase. Moreover, the composition of the organic filter phase influences the selectivity of the extraction. We show that analytes can be rapidly enriched from a 50 μL donor phase at the bottom of a sample vial, via an immiscible organic filter phase, into a 2 μL acceptor phase which consists of a droplet that is hanging from a (conductive) pipet tip in the organic filter phase. Acylcarnitines spiked to human plasma as a donor phase were extracted reproducibly with good linearity and a 10-fold improved limit of detection and, importantly, resulted in a stable, protein-free nanoelectrospray signal. Finally, a proof of principle toward the online integration in an automated nanoelectrospray-direct infusion-mass spectrometry platform has been realized. This makes 3-phase electroextraction (3-phase EE) a novel sample purification and enrichment method, with straightforward online integration possibility. We envision that 3-phase EE will enable new possibilities using electrokinetic sample pretreatment for fully automated, high-throughput bioanalysis purposes

Three-Phase Electroextraction: A New (Online) Sample Purification and Enrichment Method for Bioanalysis

The migration and at the same time enrichment of analytes from a liquid aqueous sample donor phase through an immiscible organic solvent layer acting as a filter phase into a liquid aqueous acceptor phase is enabled by the application of an electric field between the donor and acceptor phase. The organic filter phase acts as a purification filter, which prevents, for example, proteins from migrating into the acceptor phase. Moreover, the composition of the organic filter phase influences the selectivity of the extraction. We show that analytes can be rapidly enriched from a 50 μL donor phase at the bottom of a sample vial, via an immiscible organic filter phase, into a 2 μL acceptor phase which consists of a droplet that is hanging from a (conductive) pipet tip in the organic filter phase. Acylcarnitines spiked to human plasma as a donor phase were extracted reproducibly with good linearity and a 10-fold improved limit of detection and, importantly, resulted in a stable, protein-free nanoelectrospray signal. Finally, a proof of principle toward the online integration in an automated nanoelectrospray-direct infusion-mass spectrometry platform has been realized. This makes 3-phase electroextraction (3-phase EE) a novel sample purification and enrichment method, with straightforward online integration possibility. We envision that 3-phase EE will enable new possibilities using electrokinetic sample pretreatment for fully automated, high-throughput bioanalysis purposes

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

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

Flavor Profiling Using Comprehensive Mass Spectrometry Analysis of Metabolites in Tomato Soups

Trained sensory panels are regularly used to rate food products but do not allow for data-driven approaches to steer food product development. This study evaluated the potential of a molecular-based strategy by analyzing 27 tomato soups that were enhanced with yeast-derived flavor products using a sensory panel as well as LC-MS and GC-MS profiling. These data sets were used to build prediction models for 26 different sensory attributes using partial least squares analysis. We found driving separation factors between the tomato soups and metabolites predicting different flavors. Many metabolites were putatively identified as dipeptides and sulfur-containing modified amino acids, which are scientifically described as related to umami or having &ldquo;garlic-like&rdquo; and &ldquo;onion-like&rdquo; attributes. Proposed identities of high-impact sensory markers (methionyl-proline and asparagine-leucine) were verified using MS/MS. The overall results highlighted the strength of combining sensory data and metabolomics platforms to find new information related to flavor perception in a complex food matrix

Flavor Profiling Using Comprehensive Mass Spectrometry Analysis of Metabolites in Tomato Soups

Trained sensory panels are regularly used to rate food products but do not allow for data-driven approaches to steer food product development. This study evaluated the potential of a molecular-based strategy by analyzing 27 tomato soups that were enhanced with yeast-derived flavor products using a sensory panel as well as LC-MS and GC-MS profiling. These data sets were used to build prediction models for 26 different sensory attributes using partial least squares analysis. We found driving separation factors between the tomato soups and metabolites predicting different flavors. Many metabolites were putatively identified as dipeptides and sulfur-containing modified amino acids, which are scientifically described as related to umami or having “garlic-like” and “onion-like” attributes. Proposed identities of high-impact sensory markers (methionyl-proline and asparagine-leucine) were verified using MS/MS. The overall results highlighted the strength of combining sensory data and metabolomics platforms to find new information related to flavor perception in a complex food matrix

Flavor Profiling Using Comprehensive Mass Spectrometry Analysis of Metabolites in Tomato Soups

Trained sensory panels are regularly used to rate food products but do not allow for data-driven approaches to steer food product development. This study evaluated the potential of a molecular-based strategy by analyzing 27 tomato soups that were enhanced with yeast-derived flavor products using a sensory panel as well as LC-MS and GC-MS profiling. These data sets were used to build prediction models for 26 different sensory attributes using partial least squares analysis. We found driving separation factors between the tomato soups and metabolites predicting different flavors. Many metabolites were putatively identified as dipeptides and sulfur-containing modified amino acids, which are scientifically described as related to umami or having “garlic-like” and “onion-like” attributes. Proposed identities of high-impact sensory markers (methionyl-proline and asparagine-leucine) were verified using MS/MS. The overall results highlighted the strength of combining sensory data and metabolomics platforms to find new information related to flavor perception in a complex food matrix