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.

Metabolomics of Human Semen: A Review of Different Analytical Methods to Unravel Biomarkers for Male Fertility Disorders.

BACKGROUND: Human life without sperm is not possible. Therefore, it is alarming that the fertilizing ability of human spermatozoa is continuously decreasing. The reasons for that are widely unknown, but there is hope that metabolomics-based investigations may be able to contribute to overcoming this problem. This review summarizes the attempts made so far. METHODS: We will discuss liquid chromatography-mass spectrometry (LC-MS), gas chromatography (GC), infrared (IR) and Raman as well as nuclear magnetic resonance (NMR) spectroscopy. Almost all available studies apply one of these methods. RESULTS: Depending on the methodology used, different compounds can be detected, which is (in combination with sophisticated methods of bioinformatics) helpful to estimate the state of the sperm. Often, but not in all cases, there is a correlation with clinical parameters such as the sperm mobility. CONCLUSIONS: LC-MS detects the highest number of metabolites and can be considered as the method of choice. Unfortunately, the reproducibility of some studies is poor, and, thus, further improvements of the study designs are needed to overcome this problem. Additionally, a stronger focus on the biochemical consequences of the altered metabolite concentrations is also required.

Insights into structure, affinity, specificity, and function of GAG-protein interactions through the chemoenzymatic preparation of defined sulfated oligohyaluronans.

High amounts of glycosaminoglycans (GAG) such as hyaluronan (HA) occur in connective tissues. There is nowadays increasing evidence that a "sulfation code" exists which mediates numerous GAG functions. High molecular weight and inhomogeneity of GAG, however, aggravated detailed studies. Thus, synthetic oligosaccharides were urgently required. We will review here chemoenzymatic and analytic strategies to provide defined sulfated and anomerically modified GAG oligosaccharides of the HA type. Representative studies of protein/GAG interactions by (bio)chemical and biophysical methods are reported yielding novel insights into GAG-protein binding. Finally, the biological conclusions and in vivo applications of defined sulfated GAG oligosaccharides will be discussed.

Chemically modified glycosaminoglycan derivatives as building blocks for biomaterial coatings and hydrogels.

Tissue regeneration is regulated by the cellular microenvironment, e.g. the extracellular matrix. Here, sulfated glycosaminoglycans (GAG), are of vital importance interacting with mediator proteins and influencing their biological activity. Hence, they are promising candidates for controlling tissue regeneration. This review addresses recent achievements regarding chemically modified GAG as well as collagen/GAG-based coatings and hydrogels including (i) chemical functionalization strategies for native GAG, (ii) GAG-based biomaterial strategies for controlling cellular responses, (iii) (bio)chemical methods for characterization and iv) protein interaction profiles and attained tissue regeneration in vitro and in vivo. The potential of GAG for bioinspired, functional biomaterials is highlighted.

Differences in the sperm metabolomes of smoking and nonsmoking men†.

Currently, spermiogram analysis is the most relevant method used to clarify the potential infertility of a couple. However, in some cases, the reasons for infertility remain obscure. Smoking is among the factors that have been described to adversely affect male fertility. Smoking increases oxidative stress and thus promotes various pathological processes. Comparative studies, particularly those on metabolomic changes in sperm and seminal plasma caused by smoking, have not yet been published. Thus, the present pilot study aimed at the mass spectrometric characterization of the metabolomes of specimens from both smoking and nonsmoking subjects and the comparison of the evaluated data in terms of sperm apoptosis and spermiogram parameters. The results provided evidence that the conventional spermiogram is not altered in smokers compared to nonsmokers. However, a more careful investigation of sperm cells by metabolomic profiling reveals profound effects of smoking on sperm: first, nitrogen oxide synthase, a marker of oxidative stress, is activated. Second, the uptake of fatty acids into sperm mitochondria is reduced, leading to an impaired energy supply. Third, phenylalanine hydroxylation and tryptophan degradation, which are both indications of altered tetrahydrobiopterin biosynthesis, are reduced. Moreover, flow cytometry approaches indicated increased sperm caspase-3 activity, a sign of apoptosis. The present study clearly shows the negative effects of smoking on semen quality. Especially for idiopathic cases, metabolomic profiling can help to shed light on male subfertility or infertility.

Reduced lipolysis in lipoma phenocopies lipid accumulation in obesity.

BACKGROUND: Elucidation of lipid metabolism and accumulation mechanisms is of paramount importance to understanding obesity and unveiling therapeutic targets. In vitro cell models have been extensively used for these purposes, yet, they do not entirely reflect the in vivo setup. Conventional lipomas, characterized by the presence of mature adipocytes and increased adipogenesis, could overcome the drawbacks of cell cultures. Also, they have the unique advantage of easily accessible matched controls in the form of subcutaneous adipose tissue (SAT) from the same individual. We aimed to determine whether lipomas are a good model to understand lipid accumulation. METHODS: We histologically compared lipomas and control SAT, followed by assessment of the lipidome using high-resolution 1H NMR spectroscopy and ESI-IT mass spectrometry. RNA-sequencing was used to obtain the transcriptome of lipomas and the matched SAT. RESULTS: We found a significant increase of small-size (maximal axis < 70 µm) and very big (maximal axis > 150 µm) adipocytes within lipomas. This suggests both enhanced adipocyte proliferation and increased lipid accumulation. We further show that there is no significant change in the lipid composition compared to matched SAT. To better delineate the pathophysiology of lipid accumulation, we considered two groups with different genetic backgrounds: (1) lipomas with HMGA2 fusions and (2) without gene fusions. To reduce the search space for genes that are relevant for lipid pathophysiology, we focused on the overlapping differentially expressed (DE) genes between the two groups. Gene Ontology analysis revealed that DE genes are enriched in pathways related to lipid accumulation. CONCLUSIONS: We show that the common shared lipid accumulation mechanism in lipoma is a reduction in lipolysis, with most gene dysregulations leading to a reduced cAMP in the adipocyte. Superficial lipomas could thus be used as a model for lipid accumulation through altered lipolysis as found in obese patients.

The repertoire of Adhesion G protein-coupled receptors in adipocytes and their functional relevance.

BACKGROUND: G protein-coupled receptors (GPCR) are well-characterized regulators of a plethora of physiological functions among them the modulation of adipogenesis and adipocyte function. The class of Adhesion GPCR (aGPCR) and their role in adipose tissue, however, is poorly studied. With respect to the demand for novel targets in obesity treatment, we present a comprehensive study on the expression and function of this enigmatic GPCR class during adipogenesis and in mature adipocytes. METHODS: The expression of all aGPCR representatives was determined by reanalyzing RNA-Seq data and by performing qPCR in different mouse and human adipose tissues under low- and high-fat conditions. The impact of aGPCR expression on adipocyte differentiation and lipid accumulation was studied by siRNA-mediated knockdown of all expressed members of this receptor class. The biological characteristics and function of mature adipocytes lacking selected aGPCR were analyzed by mass spectrometry and biochemical methods (lipolysis, glucose uptake, adiponectin secretion). RESULTS: More than ten aGPCR are significantly expressed in visceral and subcutaneous adipose tissues and several aGPCR are differentially regulated under high-caloric conditions in human and mouse. Receptor knockdown of six receptors resulted in an impaired adipogenesis indicating their expression is essential for proper adipogenesis. The altered lipid composition was studied in more detail for two representatives, ADGRG2/GPR64 and ADGRG6/GPR126. While GPR126 is mainly involved in adipocyte differentiation, GPR64 has an additional role in mature adipocytes by regulating metabolic processes. CONCLUSIONS: Adhesion GPCR are significantly involved in qualitative and quantitative adipocyte lipid accumulation and can control lipolysis. Factors driving adipocyte formation and function are governed by signaling pathways induced by aGPCR yielding these receptors potential targets for treating obesity.

Different glycolipids in sperm from different freshwater fishes - A high-performance thin-layer chromatography/electrospray ionization mass spectrometry study.

RATIONALE: Glycolipids play important roles in many physiological processes - despite their commonly low abundance. This study summarizes selected data on the (glyco)lipid composition of sperm from different fish species. METHODS: Lipid extraction of fish sperm was performed according to the procedure by Bligh and Dyer. The lipid composition of the organic extracts was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization ion trap (ESI-IT)MS coupled to high-performance thin-layer chromatography (HPTLC). RESULTS: It was shown that sperm from carp, northern pike, rainbow trout and burbot contain high amounts of neutral and acidic glycosphingolipids as well as sulfoglycolipids. These particular lipids are presumably involved in reproduction requirements. CONCLUSIONS: Phospholipids and glycolipids in crude lipid extracts can be analyzed in parallel by MS coupled to TLC. The direct application of tandem mass spectrometry (MS/MS) helps to elucidate the glycolipid structure. Thus, compositional analysis can be performed very rapidly.

Increased plasma phosphatidylcholine/lysophosphatidylcholine ratios in patients with Parkinson's disease.

RATIONALE: Changes in lipid composition might be associated with the onset and progression of various neurodegenerative diseases. Herein, we investigated the changes in the plasma phosphatidylcholine (PC)/lysophosphatidylcholine (LPC) ratios in patients with Parkinson's disease (PD) in comparison with healthy subjects and their correlation with clinico-pathological features. METHODS: The study included 10 controls and 25 patients with PD. All patients were assigned to groups based on clinico-pathological characteristics (gender, age at examination, duration of disease and Hoehn and Yahr (H&Y) stage). The analysis of the PC/LPC intensity ratios in plasma lipid extracts was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: PD patients exhibited an increased PC/LPC intensity ratio in comparison with the control group of healthy subjects. Furthermore, the investigated ratio was shown to be correlated with clinico-pathological parameters, in particular with H&Y stage and disease duration. The PC/LPC intensity ratio in plasma samples of PD patients was found to be elevated in all examined H&Y stages and throughout the disease duration. CONCLUSIONS: To our knowledge, this is the first study examining the PC/LPC ratios in plasma of patients with PD and illustrating their correlation with clinico-pathological features. Although the presented results may be considered as preliminary due to the limited number of participants, the observed alterations of PC/LPC ratios in plasma might be a first step in the characterization of plasma lipid changes in PD patients and an indicator of lipid reconfiguration.

Differences in the lipid patterns during maturation of 3T3-L1 adipocytes investigated by thin-layer chromatography, gas chromatography, and mass spectrometric approaches.

Populations of industrialized countries have registered a dramatically increasing prevalence in obesity for many years. Despite continuous research, mechanisms involved in the storage and utilization of chemical energy in adipocytes are still under investigation. Adipocytes have the task to store excessive energy in the form of triacylglycerols (TG) and it is already well-known that the fatty acyl composition of TG is largely determined by the composition of the diet. In contrast to TG, the composition of adipocyte phospholipids was less comprehensively investigated. In this study, the compositions of the most abundant phospholipid classes of 3T3-L1 undifferentiated (preadipocytes) and differentiated cells (adipocytes) were determined. The lipid fractions were isolated by normal phase high-performance thin-layer chromatography and subsequently analyzed by electrospray ionization mass spectrometry. Additionally, the fatty acyl (FA) compositions were determined by gas chromatography. The positions of the FA residues were additionally confirmed by phospholipase A2 digestion. The advantages and disadvantages of the different analytical approaches will be discussed. It will be shown that undifferentiated 3T3-L1 and mature adipocytes differ extremely regarding their compositions. This goes along with an increase in odd-chain fatty acids. Graphical abstract.

The lipid head group is the key element for substrate recognition by the P4 ATPase ALA2: a phosphatidylserine flippase.

Type IV P-type ATPases (P4 ATPases) are lipid flippases that catalyze phospholipid transport from the exoplasmic to the cytoplasmic leaflet of cellular membranes, but the mechanism by which they recognize and transport phospholipids through the lipid bilayer remains unknown. In the present study, we succeeded in purifying recombinant aminophospholipid ATPase 2 (ALA2), a member of the P4 ATPase subfamily in Arabidopsis thaliana, in complex with the ALA-interacting subunit 5 (ALIS5). The ATP hydrolytic activity of the ALA2-ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine. Small changes in the stereochemistry or the functional groups of the phosphatidylserine head group affected enzymatic activity, whereas alteration in the length and composition of the acyl chains only had minor effects. Likewise, the enzymatic activity of the ALA2-ALIS5 complex was stimulated by both mono- and di-acyl phosphatidylserines. Taken together, the results identify the lipid head group as the key structural element for substrate recognition by the P4 ATPase.

Phospholipase A2 products predict the hematopoietic support capacity of horse serum.

Horse serum is commonly used as an additive to support the maintenance of hematopoietic progenitor cells in culture. However, the wide variability in the performance of different lots calls for parallel testing of multiple batches over extended periods of culture. Identification of the serum components that determine hematopoietic support would therefore save considerable time and effort and would help to standardize culture procedures. We report here that the ability of horse serum to support the self-renewal of multipotent murine hematopoietic progenitor FDCP-Mix cells is correlated to the concentration of specific fatty acid products of phospholipase A2 and more closely to the spectrum of eicosanoids generated by their further processing through cyclooxygenase and lipoxygenase pathways. Supportive sera have low levels of lysophosphatidylcholine and inflammatory eicosanoids. This links known markers of inflammation, infection and platelet activation to the ability of serum to maintain progenitor cells in an undifferentiated state, providing a means for prospective identification of suitable sera as well as quality control of the production process.

Assisted reproduction for felid species conservation-Sperm competences at risk.

Cryobanking of gametes in combination with artificial insemination is an essential option to support conservation programmes for endangered and threatened species. About two-thirds of the felid species are classified as 'near threatened', 'vulnerable' or 'endangered' (www.cites.org), and mostly, epididymal sperm are collected from euthanized or castrated male felids and cryopreserved. However, epididymal compared with ejaculated and cryopreserved compared with fresh sperm have a limited potential to fertilize if vaginal non-surgical insemination is applied in feline species. Missing or highly diluted seminal fluid in epididymal and cryopreserved sperm, as well as a potential interference of extender ingredients with the natural interactive properties of sperm in the female genital tract is discussed as potential drawback which hampers a proper sperm transit and fertilization besides the limited longevity of cryopreserved feline sperm. Individual components in seminal fluid as well as cryoextenders may adversely alter sperm properties and have a different impact on fertility and preservation success. The identification and investigation of beneficial as well as detrimental components is a precondition to deduce options for improving the process of cryopreservation in felids, particularly, if only epididymal sperm are available.

Normal-phase versus reversed-phase thin-layer chromatography (TLC) to monitor oxidized phosphatidylcholines by TLC/mass spectrometry.

RATIONALE: Normal-phase thin-layer chromatography (NP-TLC) is an established method for the separation of all major phospholipid classes according to the different polarities of the head groups. In contrast, reversed-phase (RP)-TLC is much less frequently used for this purpose. This study aimed to compare the NP and the RP approach regarding their separation potential of phospholipid oxidation products. METHODS: Commercially available 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC) (PC 16:0/18:1) and 1-palmitoyl-2-linoleoyl-sn-phosphatidylcholine (PLPC) (PC 16:0/18:2) were oxidized by NaMnO4 . Oxidation products were subsequently separated by NP- and RP-TLC and analyzed by electrospray ionization mass spectrometry. RESULTS: In comparison with NP-TLC, RP-TLC was clearly superior regarding the separation of oxidation products of phospholipids. RP-TLC enabled the separation not only of primary oxidation products of POPC such as alcohols and ketones but also of secondary oxidation products. Furthermore some oxidation products, such as aldehydes, were only detectable by ESI after RP-TLC but not after NP-TLC. CONCLUSIONS: RP-TLC is the method of choice to characterize oxidized PL such as oxidized phosphatidylcholines.

Short-chain lipid-conjugated pH sensors for imaging of transporter activities in reconstituted systems and living cells.

The design of ion sensors has gained importance for the study of ion dynamics in cells, with fluorescent proton nanosensors attracting particular interest because of their applicability in monitoring pH gradients in biological microcompartments and reconstituted membrane systems. In this work, we describe the improved synthesis, photophysical properties and applications of pH sensors based on amine-reactive pHrodo esters and short-chain lipid derivatives of phosphoethanolamine. The major features of these novel probes include strong fluorescence under acidic conditions, efficient partitioning into membranes, and extractability by back exchange to albumin. These features allow for the selective labeling of the inner liposomal leaflet in reconstituted membrane systems for studying proton pumping activities in a quantitative fashion, as demonstrated by assaying the activity of a plant plasma membrane H+-ATPase. Furthermore, the short-chain lipid-conjugated pH sensors enable the monitoring of pH changes from neutral to acidic conditions in the endocytic pathway of living cells. Collectively, our results demonstrate the applicability of short-chain lipid-conjugated sensors for in vivo and in vitro studies and thus pave the way for the design of lipid-conjugated sensors selective to other biologically relevant ions, e.g. calcium and sodium.

Automated semen analysis by SQA Vision® versus the manual approach-A prospective double-blind study.

Next to clinical investigations, the evaluation of male fertility relies mainly on detailed sperm analyses, for example, cell counting, motility, cell morphology and vitality testing. The manual creation of a spermiogram is time- and material-consuming. Therefore, reliable high-throughput systems that may be substituted for manual methods are urgently needed. The present study aimed to compare conventional sperm analysis performed as per WHO 5th guidelines and semen analysis performed with the SQA Vision® machine. SQA Vision® is a commercial device for automated sperm analysis. Data obtained independently by both methods were compared by statistical analyses using Bland-Altman plots and Passing-Bablok regression analyses. The analyses revealed that the results for sperm concentration and total motility were comparable. The agreement for progressive motility was poor, and there were clear deviations in the determination of normal sperm morphology. Passing-Bablok regression analyses and the consideration of the 95% confidence intervals pointed out systematic and proportional differences between the manual semen analysis and the automated approach.

Nanomechanics of Extracellular Vesicles Reveals Vesiculation Pathways.

Extracellular vesicles (EVs) are emerging as important mediators of cell-cell communication as well as potential disease biomarkers and drug delivery vehicles. However, the mechanical properties of these vesicles are largely unknown, and processes leading to microvesicle-shedding from the plasma membrane are not well understood. Here an in depth atomic force microscopy force spectroscopy study of the mechanical properties of natural EVs is presented. It is found that several natural vesicles of different origin have a different composition of lipids and proteins, but similar mechanical properties. However, vesicles generated by red blood cells (RBC) at different temperatures/incubation times are different mechanically. Quantifying the lipid content of EVs reveals that their stiffness decreases with the increase in their protein/lipid ratio. Further, by maintaining RBC at "extreme" nonphysiological conditions, the cells are pushed to utilize different vesicle generation pathways. It is found that RBCs can generate protein-rich soft vesicles, possibly driven by protein aggregation, and low membrane-protein content stiff vesicles, likely driven by cytoskeleton-induced buckling. Since similar cortical cytoskeleton to that of the RBC exists on the membranes of most mammalian cells, our findings help advancing the understanding of the fundamental process of vesicle generation.

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.

The combination of 2,5-dihydroxybenzoic acid and 2,5-dihydroxyacetophenone matrices for unequivocal assignment of phosphatidylethanolamine species in complex mixtures.

Unequivocal assignment of phospholipid peaks in complex mixtures is difficult if only the m/z values but no tandem mass spectrometry (MS/MS) data are available. This is usually the case for matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) MS imaging experiments and the analysis has normally to be performed without prior separation. Another problem might be the often matrix-induced loss of one methyl group in phosphatidylcholine (PC) species, which makes them detectable as negative ions becoming isomers of some phosphatidylethanolamines (PEs). Selected lipid mixtures of known compositions were investigated by negative ion MALDI-TOF MS and various imaging experiments. In addition to common matrices such as 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9-AA), different binary matrices, including 2,5-dihydroxyacetophenone (2,5-DHAP) as matrix additive to DHB, were tested to probe their performance in both ionization modes. Beside artificial PC and PE mixtures of known compositions, egg yolk and liver extracts as well as cryosections from liver and pancreas tissue were selected as biologically relevant systems. The majority of the binary MALDI matrices used here leads to the loss of a methyl group from PC in the negative ion mode, which makes the clear identification of PE species ambiguous. However, this problem does not apply if a mixture of DHB and 2,5-DHAP is used. Therefore, the application of DHB/2,5-DHAP as matrix is a simple method to unequivocally identify PEs even in complex mixtures and tissue sections as negative ions and without the necessity to separate the individual lipid classes prior to MS detection. Graphical abstract Many common MALDI matrices (such as 9-AA) induce the loss of a methyl group from PC rendering the PC detectable as negative ion. These ions (m/z 744.6 in the upper trace) represent isomers of typical PE species. It will be shown that this problem can be avoided if mixtures between DHB and 2,5-DHAP are applied. At these conditions, POPC is exclusively detectable as a matrix adduct with DHB (at m/z 912.6, lower trace) and does not interfere with PE. This approach can also be used in MALDI MS imaging.

Towards Understanding Male Infertility After Spinal Cord Injury Using Quantitative Proteomics.

The study of male infertility after spinal cord injury (SCI) has enhanced the understanding of seminal plasma (SP) as an important regulator of spermatozoa function. However, the most important factors leading to the diminished sperm motility and viability observed in semen of men with SCI remained unknown. Thus, to explore SP related molecular mechanisms underlying infertility after SCI, we used mass spectrometry-based quantitative proteomics to compare SP retrieved from SCI patients to normal controls. As a result, we present an in-depth characterization of the human SP proteome, identifying ∼2,800 individual proteins, and describe, in detail, the differential proteome observed in SCI. Our analysis demonstrates that a hyper-activation of the immune system may influence some seminal processes, which likely are not triggered by microbial infection. Moreover, we show evidence of an important prostate gland functional failure,i.e.diminished abundance of metabolic enzymes related to ATP turnover and those secreted via prostasomes. Further we identify the main outcome related to this fact and that it is intrinsically linked to the low sperm motility in SCI. Together, our data highlights the molecular pathways hindering fertility in SCI and shed new light on other causes of male infertility.