From Oxidized Fatty Acids to Dimeric Species: In Vivo Relevance, Generation and Methods of Analysis.

The occurrence of free fatty acids (FFAs) and the generation of reactive oxygen species (ROS) such as hydroxyl radicals (HO●) or hypochlorous acid (HOCl) is characteristic of inflammatory diseases, for instance, rheumatoid arthritis. Unsaturated fatty acids react with ROS yielding a variety of important products such as peroxides and chlorohydrins as primary and chain-shortened compounds (e.g., aldehydes and carboxylic acids) as secondary products. These modified fatty acids are either released from phospholipids by phospholipases or oxidatively modified subsequent to their release. There is increasing evidence that oligomeric products are also generated upon these processes. Fatty acid esters of hydroxy fatty acids (FAHFAs) are considered as very important products, but chlorinated compounds may be converted into dimeric and (with smaller yields) oligomeric products, as well. Our review is structured as follows: first, the different types of FFA oligomers known so far and the mechanisms of their putative generation are explained. Industrially relevant products as well as compounds generated from the frying of vegetable oils are also discussed. Second, the different opinions on whether dimeric fatty acids are considered as "friends" or "foes" are discussed.

A Five-Year Update on Matrix Compounds for MALDI-MS Analysis of Lipids.

Matrix-assisted laser desorption and ionization (MALDI) is a widely used soft-ionization technique of modern mass spectrometry (MS). MALDI enables the analysis of nearly all chemical compounds-including polar and apolar (phospho)lipids-with a minimum extent of fragmentation. MALDI has some particular advantages (such as the possibility to acquire spatially-resolved spectra) and is competitive with the simultaneously developed ESI (electrospray ionization) MS. Although there are still some methodological aspects that need to be elucidated in more detail, it is obvious that the careful selection of an appropriate matrix plays the most important role in (lipid) analysis. Some lipid classes can be detected exclusively if the optimum matrix is used, and the matrix determines the sensitivity by which a particular lipid is detected within a mixture. Since the matrix is, thus, crucial for optimum results, we provide here an update on the progress in the field since our original review in this journal in 2018. Thus, only the development during the last five years is considered, and lipids are sorted according to increasing complexity, starting with free fatty acids and ending with cardiolipins and phosphoinositides.

Mass spectrometric investigations of the action of hypochlorous acid on monomeric and oligomeric components of glycosaminoglycans.

Hypochlorous acid (HOCl) is a strong non-radical oxidant, which is generated during inflammatory processes under the catalysis of the enzyme myeloperoxidase (MPO). HOCl reacts particularly with sulfhydryl and amino acid residues but affects also many other biomolecules. For instance, the glycosaminoglycans of articular cartilage and synovial fluids (such as hyaluronan) undergo degradation in the presence of HOCl at which the native polysaccharide is fragmented into oligosaccharides in a complex reaction. This is an initial mass spectrometry (MS)-based investigation dealing with the HOCl-induced degradation of glycosaminoglycans and the conversion of the related monosaccharides into chlorinated products. In particular, it will be shown that the reaction between HOCl and hyaluronan is slower than originally assumed and results in the generation of different products (particularly the hyaluronan monosaccharides) by the cleavage of the ß-1,3/1,4-glycosidic linkages. The MS detection of chlorinated products is, however, only possible in the case of the monosaccharides. Potential reasons will be discussed.

Combined Use of MALDI-TOF Mass Spectrometry and 31P NMR Spectroscopy for the Analysis of (Phospho)Lipids.

Lipids are important and abundant constituents of all biological tissues and body fluids. In particular, phospholipids (PLs) constitute a major part of the cellular membrane and play a role in signal transduction, and some selected PLs are increasingly considered as potential disease markers. Unfortunately, methods of lipid analysis are less established in comparison to techniques of protein analysis. Mass spectrometry (MS) is an increasingly used technique to analyze lipids, especially in combination with electrospray ionization MS, which is the most commonly used ionization technique in lipidomics. Matrix-assisted laser desorption/ionization coupled to time-of-flight MS (MALDI-TOF MS) has itself proven to represent a useful tool in the field of lipid analysis. 31P nuclear magnetic resonance (NMR) spectroscopy, another powerful method for PL analysis, represents a direct quantitative method and does not suffer from suppression effects.This paper gives an overview of methodological aspects of MALDI-TOF MS and 31P NMR in lipid research and summarizes the specific advantages and drawbacks of both methods. In particular, suppression effects in MS will be highlighted, and possible ways to overcome this problem, e.g., the use of different matrices and separation of the relevant lipid mixture prior to analysis, will be discussed.

A new update of MALDI-TOF mass spectrometry in lipid research.

Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS) is an indispensable tool in modern lipid research since it is fast, sensitive, tolerates sample impurities and provides spectra without major analyte fragmentation. We will discuss some methodological aspects, the related ion-forming processes and the MALDI MS characteristics of the different lipid classes (with the focus on glycerophospholipids) and the progress, which was achieved during the last ten years. Particular attention will be given to quantitative aspects of MALDI MS since this is widely considered as the most serious drawback of the method. Although the detailed role of the matrix is not yet completely understood, it will be explicitly shown that the careful choice of the matrix is crucial (besides the careful evaluation of the positive and negative ion mass spectra) in order to be able to detect all lipid classes of interest. Two developments will be highlighted: spatially resolved Imaging MS is nowadays well established and the distribution of lipids in tissues merits increasing interest because lipids are readily detectable and represent ubiquitous compounds. It will also be shown that a combination of MALDI MS with thin-layer chromatography (TLC) enables a fast spatially resolved screening of an entire TLC plate which makes the method competitive with LC/MS.

Phospholipid Profiles for Phenotypic Characterization of Adipose-Derived Multipotent Mesenchymal Stromal Cells.

Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LC-MS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.

De novo synthesis of phospholipids and sphingomyelin in multipotent stromal cells - Monitoring studies by mass spectrometry.

Musculoskeletal diseases are extremely widespread and a significant burden on the health systems of the industrialized countries. The use of mesenchymal stromal cells is a promising approach to cure cartilage and tendon injuries, which often also occur in younger people as consequences of sport accidents. Although particular interest is on the collagen and the glycosaminoglycan composition of the tendon and potential alterations compared to healthy tissue, there is nowadays also increasing evidence that some selected phospholipids (PL) are potential mediators of tissue regeneration. Therefore, PL (and potential changes thereof) attract increasing interest in this field. We have used positive and negative ion matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to elucidate the lipid compositions of human mesenchymal stromal cells in dependence on the composition of the cell culture medium and the cultivation time. The de novo biosynthesis of PL was monitored by adding 13C labeled glucose or deuterated palmitic acid (d31-PA) to the cells and the incorporation of 13C or 2H into the different PL classes was investigated by electrospray ionization (ESI) mass spectrometry (MS). It is remarkable that all PL classes (for instance, phosphatidylcholine and -inositol) exhibited 13C incorporation - but not the sphingomyelin (SM) which is the most abundant sphingolipid in the majority of human tissues and body fluids. Using suitable internal standards it could be shown, that only 12C-containing SM is de novo generated while no 13C-labeled SM could be monitored - independent of the cultivation time, which was varied between 7 and 28 days. SM impurities stemming from the cell culture medium and the used MALDI matrix compounds (2,5-dihydroxybenzoic acid (DHB) or 9-aminoacridine (9-AA)) could be ruled out. However, incorporation of deuterated palmitic acid (d31-PA) could be observed for multiple PL, including SM. Therefore, it is suggested that there must exist another, so far unknown SM biosynthesis pathway. This pathway does not make use of glucose but relies on the use of other molecules as energy sources. Potential pathways to explain the experimental observations are discussed.

Free cholesterol, cholesterol precursor and plant sterol levels in atherosclerotic plaques are independently associated with symptomatic advanced carotid artery stenosis.

BACKGROUND AND AIMS: Circulating sterols result either from cholesterol (CH) synthesis or intestinal uptake. They are mainly esterified and can be oxygenated. Sterols accumulate in atherosclerotic plaques whereby their clinical impact is uncertain. Here, we determined associations between circulating and plaque sterol levels in patients with advanced carotid artery stenosis in respect to a prior ischemic event and statin treatment. METHODS: Free and esterified CH, CH precursors and plant sterols as well as oxysterols were quantified by liquid chromatography-tandem mass spectrometry in 63 consecutive patients undergoing carotid endarterectomy. RESULTS: CH, CH precursors, plant sterols and oxysterols accumulated in carotid artery plaques. Absolute circulating sterol levels were not predictive for their corresponding plaque levels. After normalisation to CH, plant sterol but not oxysterol levels correlated between plasma and plaques. Among the circulating sterols, oxysterols occurred proportionally less in plaques. Furthermore, CH and plant sterols were less esterified in plaques than in plasma. Patients who experienced a prior ischemic event (n = 29) and asymptomatic patients had, except for lanosterol, comparable circulating sterol levels. In contrast, the absolute plaque levels of free CH, CH precursors and plant sterols as well as oxysterols were increased in symptomatic compared to asymptomatic patients. These differences remained significant for free CH, precursors and 3 out of 4 analyzed plant sterols after adjustment to the most influencing covariates - statin treatment, type 2 diabetes and age. CONCLUSIONS: Increased absolute plaque levels of free CH, precursors and plant sterols predict an ischemic event in patients with advanced carotid artery stenosis.

Recent Developments of Useful MALDI Matrices for the Mass Spectrometric Characterization of Lipids.

Matrix-assisted laser desorption/ionization (MALDI) is one of the most successful "soft" ionization methods in the field of mass spectrometry and enables the analysis of a broad range of molecules, including lipids. Although the details of the ionization process are still unknown, the importance of the matrix is commonly accepted. Both, the development of and the search for useful matrices was, and still is, an empirical process, since properties like vacuum stability, high absorption at the laser wavelength, etc. have to be fulfilled by a compound to become a useful matrix. This review provides a survey of successfully used MALDI matrices for the lipid analyses of complex biological samples. The advantages and drawbacks of the established organic matrix molecules (cinnamic or benzoic acid derivatives), liquid crystalline matrices, and mixtures of common matrices will be discussed. Furthermore, we will deal with nanocrystalline matrices, which are most suitable to analyze small molecules, such as free fatty acids. It will be shown that the analysis of mixtures and the quantitative analysis of small molecules can be easily performed if the matrix is carefully selected. Finally, some basic principles of how useful matrix compounds can be "designed" de novo will be introduced.

Visualizing phosphatidylcholine via mass spectrometry imaging: relevance to human health.

INTRODUCTION: Mass spectrometry imaging (MSI) techniques are nowadays widely used to obtain spatially resolved metabolite information from biological tissues. Since (phospho)lipids occur in all animal tissues and are very sensitively detectable, they are often in the focus of such studies. This particularly applies for phosphatidylcholines (PC) which are very sensitively detectable as positive ions due to the permanent positive charge of their choline headgroup. Areas covered: After a short introduction of lipid species occurring in biological systems and approaches normally used to obtain spatially resolved mass spectra (with the focus on matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF) MSI) a survey will be given which diseases have so far been characterized by changes of the PC composition. Expert commentary: Since PC species are very sensitively detectable by MS, sensitivity is not a major issue. However, spatial resolution is still limited and cellular dimensions can be hardly resolved by MALDI-TOF MSI, which is a critical point of the available approaches. Due to lacks of reproducibility and standardization further development is required.