Scavenger Receptor BI Deficiency in Mice Is Associated With Plasma Ceramide and Sphingomyelin Accumulation and a Reduced Cholesteryl Ester Fatty Acid Length and Unsaturation Degree.

Objective: Scavenger receptor class B type I (SR-BI) is primarily known for its role in the selective uptake of cholesteryl esters (CEs) from high-density lipoproteins (HDLs). Here we investigated whether SR-BI deficiency is associated with other potentially relevant changes in the plasma lipidome than the established effect of HDL-cholesterol elevation. Methods: Targeted ultra-high-performance liquid chromatography-tandem mass spectrometry was utilized to measure lipid species in plasma from female wild-type and SR-BI knockout mice. Results: SR-BI deficiency was associated with a reduction in the average CE fatty acid length (-2%; p<0.001) and degree of CE fatty acid unsaturation (-18%; p<0.001) due to a relative shift from longer, polyunsaturated CE species CE (20:4), CE (20:5), and CE (22:6) towards the mono-unsaturated CE (18:1) species. Sphingomyelin (SM) levels were 64% higher (p<0.001) in SR-BI knockout mice without a parallel change in (lyso)phosphatidylcholine (LPC) concentrations, resulting in an increase in the SM/LPC ratio from 0.102±0.005 to 0.163±0.003 (p<0.001). In addition, lower LPC lengths (-5%; p<0.05) and fatty acid unsaturation degrees (-20%; p<0.01) were detected in SR-BI knockout mice. Furthermore, SR-BI deficiency was associated with a 4.7-fold increase (p<0.001) in total plasma ceramide (Cer) levels, with a marked >9-fold rise (p<0.001) in Cer (d18:1/24:1) concentrations. Conclusion: We have shown that SR-BI deficiency in mice not only impacts the CE concentrations, length, and saturation index within the plasma compartment, but is also associated with plasma accumulation of several Cer and SM species that may contribute to the development of specific hematological and metabolic (disease) phenotypes previously detected in SR-BI knockout mice.

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.

An automated online three-phase electro-extraction setup with machine-vision process monitoring hyphenated to LC-MS analysis.

Sample preparation is a labor-intensive and time-consuming procedure, especially for the bioanalysis of small-volume samples with low-abundant analytes. To minimize losses and dilution, sample preparation should ideally be hyphenated to downstream on-line analysis such as liquid chromatography-mass spectrometry (LC-MS). In this study, an automated three-phase electro-extraction (EE) method coupled to machine vision was developed, integrated with a robotic autosampler hyphenated to LC-MS. Eight model compounds, i.e. amitriptyline, clemastine, clomipramine, haloperidol, loperamide, propranolol, oxeladin, and verapamil were utilized for the optimization and evaluation of the automated EE setup. The stability of automated EE was evaluated by monitoring the acceptor droplet size by machine vision and recording the current during EE. A Design of Experiment approach (Box-Behnken design) was utilized to optimize the critical parameters of the EE method, i.e., the ratio of formic acid in the sample to acceptor phase, extraction voltage, and extraction time. The developed quadratic models showed good fitness (p < 0.001, R2 > 0.95). Automated EE could be achieved in less than 2 min with enrichment factors (EF) up to 387 and extraction recoveries (ER) up to 97% for academic samples. Finally, the optimized EE method was successfully applied to both spiked human urine and plasma samples with low-concentration (50 ng mL-1) analytes and a low starting sample volume of 20 µL of plasma and urine in 10-fold diluted samples. The developed automated EE setup is easy to operate, provides a fast extraction method for analytes from volume-limited biological samples, and is hyphenated with on-line LC-MS analysis. Therefore, this method can provide fast and automated sample preparation to solve bottlenecks in high-throughput bioanalysis workflows.

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.

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.

Development of a fast, online three-phase electroextraction hyphenated to fast liquid chromatography-mass spectrometry for analysis of trace-level acid pharmaceuticals in plasma.

Sample preparation is a challenge for high-throughput analysis, especially for volume-limited samples with low-abundant analytes. Ideally, sample preparation enriches the analytes of interest while removing the interferents to reduce the matrix effect and improve both sensitivity and quantification. In this study, a three-phase electroextraction (EE) method hyphenated to fast online liquid chromatography-mass spectrometry (LC-MS) was developed. Four model acidic drugs of relevance for drug monitoring in plasma, i.e. naproxen, fenoprofen, flurbiprofen, and ibuprofen, were utilized for the optimization and evaluation of the method. A Design of Experiment approach (Box-Behnken design) was used to optimize the critical parameters of the method, i.e., the type of organic solvent, pH of the sample and acceptor phase, and the extraction voltage and time. Good fitness (P < 0.02, R2 > 0.95) was observed for the developed quadratic model. Extraction could be achieved in less than 2 min (115 s) with enrichment factors (EF) up to 190 and extraction recoveries (ER) up to 38% for academic samples. Additionally, the optimized three-phase EE method was successfully applied to spiked plasma samples with low-abundant (50 ng mL-1) analytes and a low sample volume of 15 µL plasma in 10-fold diluted samples. Finally, two crucial contributors to the matrix effect of three-phase EE application on plasma samples were determined. Specifically, the ion-suppression effect in the MS source was reduced by the fast LC separation, and the matrix effect during extraction was negligible for the diluted protein-precipitated plasma samples. The developed three-phase EE method is easy to operate and provides fast and online extraction of trace-level acidic analytes from volume-limited biological samples. Therefore, this method can provide a potential solution for sample-preparation bottlenecks in high-throughput bioanalysis workflows.

Profiling acidic metabolites by capillary electrophoresis-mass spectrometry in low numbers of mammalian cells using a novel chemical derivatization approach.

The simultaneous analysis of a broad range of polar ionogenic metabolites using capillary electrophoresis-mass spectrometry (CE-MS) can be challenging, as two different analytical methods are often required, that is, one for cations and one for anions. Even though CE-MS has shown to be an effective method for cationic metabolite profiling, the analysis of small anionic metabolites often results in relatively low sensitivity and poor repeatability. In this work, a novel derivatization strategy based on trimethylmethaneaminophenacetyl bromide was developed to enable CE-MS analysis of carboxylic acid metabolites using normal CE polarity (i.e., cathode in the outlet) and detection by mass spectrometry in positive ionization mode. Optimization of derivatization conditions was performed using a response surface methodology after which the optimized method (incubation time 50 min, temperature 90°C, and pH 10) was used for the analysis of carboxylic acid metabolites in extracts from HepG2 cells. For selected metabolites, detection limits were down to 8.2 nM, and intraday relative standard deviation values for replicates (n = 3) for peak areas were below 21.5%. Metabolites related to glycolysis, tricarboxylic acid cycle, and anaerobic respiration pathways were quantified in 250,000 cell lysates, and could still be detected in extracts from only 25,000 HepG2 cell lysates (∼70 cell lysates injected).

Integration of three-phase microelectroextraction sample preparation into capillary electrophoresis.

A major strength of capillary electrophoresis (CE) is its ability to inject small sample volumes. However, there is a great mismatch between injection volume (typically <100 nL) and sample volumes (typically 20-1500 µL). Electromigration-based sample preparation methods are based on similar principles as CE. The combination of these methods with capillary electrophoresis could tackle obstacles in the analysis of dilute samples. This study demonstrates coupling of three-phase microelectroextraction (3PEE) to CE for sample preparation and preconcentration of large volume samples while requiring minimal adaptation of CE equipment. In this set-up, electroextraction takes place from an aqueous phase, through an organic filter phase, into an aqueous droplet that is hanging at the capillary inlet. The first visual proof-of-concept for this set-up showed successful extraction using the cationic dye crystal violet (CV). The potential of 3PEE for bioanalysis was demonstrated by successful extraction of the biogenic amines serotonin (5-HT), tyrosine (Tyr) and tryptophan (Trp). Under optimized conditions limits of detection (LOD) were 15 nM and 33 nM for 5-HT and Tyr respectively (with Trp as an internal standard). These LODs are comparable to other similar preconcentration methods that have been reported in conjunction with CE. Good linearity (R2 > 0.9967) was observed for both model analytes. RSDs for peak areas in technical replicates, interday and intraday variability were all satisfactory, i.e., below 14%. 5-HT, Tyr and Trp spiked to human urine were successfully extracted and separated. These results underline the great potential of 3PEE as an integrated enrichment technique from biological samples and subsequent sensitive metabolomics analysis.

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.