Max Scherer

Rapid quantification of bile acids and their conjugates in serum by liquid chromatography–tandem mass spectrometry

Beside their role as lipid solubilizers, bile acids (BAs) are increasingly appreciated as signaling factors. As ligands of G-protein coupled receptors and nuclear hormone receptors BAs control their own metabolism and act on lipid and energy metabolism. To study BA function in detail, it is necessary to use methods for their quantification covering the structural diversity of this group. Here we present a simple, sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of bile acid profiles in human plasma/serum. Protein precipitation was performed in the presence of stable-isotope labeled internal standards. In contrast to previous LC-MS/MS methods, we used a reversed-phase C18 column with 1.8microm particles and a gradient elution at basic pH. This allows base line separation of 18 bile acid species (free and conjugated) within 6.5min run time and a high sensitivity in negative ion mode with limits of detection below 10nmol/L. Quantification was achieved by standard addition and calibration lines were linear in the tested range up to 28micromol/L. Validation was performed according to FDA guidelines and overall imprecision was below 11% CV for all species. The developed LC-MS/MS method for bile acid quantification is characterized by simple sample preparation, baseline separation of isobaric species, a short analysis time and provides a valuable tool for both, routine diagnostics and the evaluation of BAs as diagnostic biomarkers in large clinical studies.

A rapid and quantitative LC-MS/MS method to profile sphingolipids

Sphingolipids comprise a highly diverse and complex class of molecules that serve not only as structural components of membranes but also as signaling molecules. To understand the differential role of sphingolipids in a regulatory network, it is important to use specific and quantitative methods. We developed a novel LC-MS/MS method for the rapid, simultaneous quantification of sphingolipid metabolites, including sphingosine, sphinganine, phyto-sphingosine, di- and trimethyl-sphingosine, sphingosylphosphorylcholine, hexosylceramide, lactosylceramide, ceramide-1-phosphate, and dihydroceramide-1-phosphate. Appropriate internal standards (ISs) were added prior to lipid extraction. In contrast to most published methods based on reversed phase chromatography, we used hydrophilic interaction liquid chromatography and achieved good peak shapes, a short analysis time of 4.5 min, and, most importantly, coelution of analytes and their respective ISs. To avoid an overestimation of species concentrations, peak areas were corrected regarding isotopic overlap where necessary. Quantification was achieved by standard addition of naturally occurring sphingolipid species to the sample matrix. The method showed excellent precision, accuracy, detection limits, and robustness. As an example, sphingolipid species were quantified in fibroblasts treated with myriocin or sphingosine-kinase inhibitor. In summary, this method represents a valuable tool to evaluate the role of sphingolipids in the regulation of cell functions.

High-Throughput Analysis of Sphingosine 1-Phosphate, Sphinganine 1-Phosphate, and Lysophosphatidic Acid in Plasma Samples by Liquid Chromatography–Tandem Mass Spectrometry

BACKGROUND Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are ubiquitous lipid messengers found in the blood and most cell types. Both lysophospholipids are ligands of G protein-coupled receptors and mediate important physiological processes. Moreover, lysophospholipids are potential biomarkers for various diseases, including atherosclerosis and cancer. Because existing methodologies are of limited value for systematic evaluations of S1P and LPA in clinical studies, we developed a fast and simple quantification method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS Sphingoid base 1-phosphates and LPA species were quantified in negative-ion mode with fragments of m/z 79 and 153, respectively. The internal standards LPA 17:0 and [(13)C(2)D(2)]S1P were added before butanol extraction. Application of hydrophilic-interaction chromatography allowed coelution of analytes and internal standards with a short analysis time of 2.5 min. RESULTS Comparison of butanol extraction with a frequently used extraction method based on strong acidification of human plasma revealed artificial formation of LPA from lysophosphatidylcholine with the latter method. Validation according to US Food and Drug Administration guidelines showed an overall imprecision (CV) of <12% and a limit of detection <6 nmol/L for all lysophospholipid species. Concentrations of S1P and sphinganine 1-phosphate (SA1P) in EDTA-containing plasma were stable for 24 h at room temperature, whereas LPA concentrations increased substantially over this period. CONCLUSIONS Our validated LC-MS/MS methodology for quantifying LPA, S1P, and SA1P features simple sample preparation and short analysis times, therefore providing a valuable tool for diagnostic evaluation of these lysophospholipids as biomarkers.

A rapid GC–MS method for quantification of positional and geometric isomers of fatty acid methyl esters

So far the most frequently used method for fatty acid (FA) analysis is GC coupled to flame ionization detector (FID). However, GC-FID does not allow profiling of FA synthesis and metabolism using stable isotopes. Here we present a rapid and sensitive GC-MS method for determination of fatty acid methyl esters (FAMEs). Fatty acid methylation was carried out by transesterification with acetyl-chloride and methanol. FAME separation applies a short and polar cyano-column resulting in an analysis time of 17.2min. Separation was achieved for positional and geometrical (cis/trans) isomers with chain lengths between C8 and C28. Partial overlap of FAMEs (e.g. for C20:2 (n-6) and C21:0) could be resolved using selected ion monitoring (SIM). The precisions for human plasma samples were better than 10% coefficient of variation (CV) except for very low abundant FAs and LODs were in the low femtomol range on column. The developed GC-MS method also allows quantification of conjugated FAs such as conjugated linoleic acid (CLA) isomers because lowering the derivatization temperature from 95 °C to room temperature prevented cis to trans double bond isomerization. Finally, profiling of fatty acid synthesis and metabolism was exemplified with stable isotope labeling of macrophages using fatty acid precursors or deuterated fatty acids. In summary, we present a fast and robust GC-MS method for fatty acid profiling of positional and geometrical isomers including CLAs as well as very long chain fatty acids (VLCFAs). The method is suitable for both clinical studies and basic research including application of stable isotope compounds.

Sphingolipid profiling of human plasma and FPLC-separated lipoprotein fractions by hydrophilic interaction chromatography tandem mass spectrometry

Sphingolipids comprise bioactive molecules which are known to play important roles both as intracellular and extracellular signalling molecules. Here we used a previously developed hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS) method to profile plasma sphingolipids. Method validation showed sufficient precision and sensitivity for application in large clinical studies. Sample stability testing demonstrated that immediate plasma separation is important to achieve reliable results. Analysis of plasma from 25 healthy blood donors revealed a comprehensive overview of free sphingoid base, sphingosylphosphorylcholine (SPC), hexosylceramide (HexCer), lactosylceramide (LacCer), and ceramide-1-phosphate (Cer1P) species level. Besides the major sphingoid base sphingosine (d18:1), we found d16:1 and d18:2 species in most of these lipid classes. Interestingly, pronounced differences were detected in the species profiles of HexCer and LacCer. Additionally, sphingolipids were quantified in lipoprotein fractions prepared by fast performance liquid chromatography (FPLC). HexCer and LacCer showed similar distributions with about 50% in LDL, 40% in HDL and less than 10% in the VLDL fraction. More than 90% of sphingoid base phosphates were found in HDL and albumin containing fractions. In summary, HILIC-MS/MS provides a valuable tool to profile minor sphingolipid species in plasma and in lipoprotein fractions. Comparing profiles from tissues or blood cells, these species profiles may help to address the origin of plasma sphingolipids.

Simultaneous Quantification of Cardiolipin, Bis(monoacylglycero)phosphate and their Precursors by Hydrophilic Interaction LC−MS/MS Including Correction of Isotopic Overlap

Cardiolipin (CL) and bis(monoacylglycero)phosphate (BMP) are unique lipid structures with important biological roles for mitochondrial integrity and endolysosomal degradation, respectively. They are synthesized from common precursors, phosphatidylglycerol (PG) and phosphatidic acid (PA). Here we present a rapid method for the simultaneous quantification of BMP, CL, PG, and PA using hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry (HILIC-MS/MS). HILIC provides coelution of lipid species and their internal standards required for accurate quantification. Coelution leads to isotope overlap of lipid species which was successfully corrected. This assay was validated in mouse heart tissue and primary human skin fibroblasts. It shows reproducibility and limits of detection sufficient for biomarker studies contributing to basic research on BMP and CL metabolism.

Specific Dietary Preferences Are Linked to Differing Gut Microbial Metabolic Activity in Response to Dark Chocolate Intake

Systems biology approaches are providing novel insights into the role of nutrition for the management of health and disease. In the present study, we investigated if dietary preference for dark chocolate in healthy subjects may lead to different metabolic response to daily chocolate consumption. Using NMR- and MS-based metabolic profiling of blood plasma and urine, we monitored the metabolic response of 10 participants stratified as chocolate desiring and eating regularly dark chocolate (CD) and 10 participants stratified as chocolate indifferent and eating rarely dark chocolate (CI) to a daily consumption of 50 g of dark chocolate as part of a standardized diet over a one week period. We demonstrated that preference for chocolate leads to different metabolic response to chocolate consumption. Daily intake of dark chocolate significantly increased HDL cholesterol by 6% and decreased polyunsaturated acyl ether phospholipids. Dark chocolate intake could also induce an improvement in the metabolism of long chain fatty acid, as noted by a compositional change in plasma fatty acyl carnitines. Moreover, a relationship between regular long-term dietary exposure to a small amount of dark chocolate, gut microbiota, and phenolics was highlighted, providing novel insights into biological processes associated with cocoa bioactives.

Stored platelets alter glycerophospholipid and sphingolipid species, which are differentially transferred to newly released extracellular vesicles.

BACKGROUND: Stored platelet concentrates (PLCs) for transfusion develop a platelet storage lesion (PSL), resulting in decreased platelet (PLT) viability and function. The processes leading to PSL have not been described in detail and no data describe molecular changes occurring in all three components of stored PLCs: PLTs, PLC extracellular vesicles (PLC‐EVs), and plasma.

Bile acid signaling after an oral glucose tolerance test

Monitoring bile acids as signal molecules in combination with a bile acid synthesis marker and the FXR regulator fibroblast growth factor 19 (FGF19), this study addresses significant postprandial changes. The efficacy of the different pathways to regulate bile acid synthesis through short heterodimer partner (SHP) dependent FXR modulation in liver, and SHP independent activation via FGF19 is demonstrated. Characteristic changes of the bile acid profile during an oral glucose tolerance test (oGTT) were investigated in 73 individuals. 15 bile acid species including conjugated and unconjugated forms were quantitatively determined with LC-MS/MS in serum samples collected at three time points during the oGTT. All conjugated bile acid species showed the same time course, a significant increase at 60 min after the glucose intake and an incline at 120 min. In contrast, a consistent decline of all unconjugated bile acids was monitored. 7α-Hydroxy-4-cholesten-3-one, an early bile acid synthesis marker, showed an inverse response with a significant decrease at 60 min which proves the efficient and rapid downregulation of CYP7A1 via FXR activation through bile acid signaling. Significantly higher levels of FGF19 were observed 120 min after glucose intake and 60 min after bile acid excursion.

The yeast acyltransferase Sct1p regulates fatty acid desaturation by competing with the desaturase Ole1p

The glycerol-3-phosphate acyltransferase Sct1p/Gat2p is shown to regulate fatty acyl chain desaturation by competing with the fatty acid desaturase Ole1p for C16:0-CoA. The activity of Sct1p depends on the level of expression and the phosphorylation state. The acyltransferase Cst26p regulates the phosphorylation of Sct1p.