Chemistry and analysis of HNE and other prominent carbonyl-containing lipid oxidation compounds

The process of lipid oxidation generates a diverse array of small aldehydes and carbonyl-containing compounds, which may occur in free form or esterified within phospholipids and cholesterol esters. These aldehydes mostly result from fragmentation of fatty acyl chains following radical oxidation, and the products can be subdivided into alkanals, alkenals (usually α,β-unsaturated), γ-substituted alkenals and bis-aldehydes. Isolevuglandins are non-fragmented di-carbonyl compounds derived from H2-isoprostanes, and oxidation of the ω−3-fatty acid docosahexenoic acid yield analogous 22 carbon neuroketals. Non-radical oxidation by hypochlorous acid can generate α-chlorofatty aldehydes from plasmenyl phospholipids. Most of these compounds are reactive and have generally been considered as toxic products of a deleterious process. The reactivity is especially high for the α,β-unsaturated alkenals, such as acrolein and crotonaldehyde, and for γ-substituted alkenals, of which 4-hydroxy-2-nonenal and 4-oxo-2-nonenal are best known. Nevertheless, in recent years several previously neglected aldehydes have been investigated and also found to have significant reactivity and biological effects; notable examples are 4-hydroxy-2-hexenal and 4-hydroxy-dodecadienal. This has led to substantial interest in the biological effects of all of these lipid oxidation products and their roles in disease, including proposals that HNE is a second messenger or signalling molecule. However, it is becoming clear that many of the effects elicited by these compounds relate to their propensity for forming adducts with nucleophilic groups on proteins, DNA and specific phospholipids. This emphasizes the need for good analytical methods, not just for free lipid oxidation products but also for the resulting adducts with biomolecules. The most informative methods are those utilizing HPLC separations and mass spectrometry, although analysis of the wide variety of possible adducts is very challenging. Nevertheless, evidence for the occurrence of lipid-derived aldehyde adducts in biological and clinical samples is building, and offers an exciting area of future research

A mass spectrometry approach for the identification and localization of small aldehyde modifications of proteins

Lipids containing polyunsaturated fatty acids are primary targets of oxidation, which produces reactive short-chain aldehydes that can covalently modify proteins, a process called lipoxidation. Improved mass spectrometry (MS) methods for the analysis of these adducts in complex biological systems are needed. Lysozyme and human serum albumin (HSA) were used as model proteins to investigate lipoxidation products formed by two short-chain aldehydes, acrolein and pentanal, which are unsaturated and saturated aldehydes respectively. The adducts formed were stabilized by NaBH4 or NaBH3CN reduction and analysed by MS. Analysis of intact modified lysozyme showed a pentanal modification resulting from Schiff's base formation (+70 Da), and up to 8 acrolein adducts, all resulting from Michael addition (+58 Da). Analysis of tryptic digests identified specific histidine, cysteine and lysine residues modified in both lysozyme and HSA, and determined characteristic amino acid-specific fragmentations. Eight different internal fragment ions were found that could be used as general diagnostic ions for pentanal- and acrolein-modified amino acids. The combined use of intact protein analysis and LC-MS/MS methods provided a powerful tool for the identification and localization of aldehyde-protein adduct, and the diagnostic ions will facilitate the development of targeted MS methods for analysis of adducts in more complex samples

BioPAN: a web-based tool to explore mammalian lipidome metabolic pathways on LIPID MAPS.

Lipidomics increasingly describes the quantification using mass spectrometry of all lipids present in a biological sample.  As the power of lipidomics protocols increase, thousands of lipid molecular species from multiple categories can now be profiled in a single experiment.  Observed changes due to biological differences often encompass large numbers of structurally-related lipids, with these being regulated by enzymes from well-known metabolic pathways.  As lipidomics datasets increase in complexity, the interpretation of their results becomes more challenging.  BioPAN addresses this by enabling the researcher to visualise quantitative lipidomics data in the context of known biosynthetic pathways.  BioPAN provides a list of genes, which could be involved in the activation or suppression of enzymes catalysing lipid metabolism in mammalian tissues

Integrated lipidomics and proteomics reveal cardiolipin alterations, upregulation of HADHA and long chain fatty acids in pancreatic cancer stem cells.

Pancreatic cancer stem cells (PCSCs) play a key role in the aggressiveness of pancreatic ductal adenocarcinomas (PDAC); however, little is known about their signaling and metabolic pathways. Here we show that PCSCs have specific and common proteome and lipidome modulations. PCSCs displayed downregulation of lactate dehydrogenase A chain, and upregulation of trifunctional enzyme subunit alpha. The upregulated proteins of PCSCs are mainly involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs. Accordingly, lipidomics reveals an increase in long and very long-chain unsaturated FAs, which are products of fatty acid elongase-5 predicted as a key gene. Moreover, lipidomics showed the induction in PCSCs of molecular species of cardiolipin with mixed incorporation of 16:0, 18:1, and 18:2 acyl chains. Our data indicate a crucial role of FA elongation and alteration in cardiolipin acyl chain composition in PCSCs, representing attractive therapeutic targets in PDAC

Evaluation of air oxidized PAPC: A multi laboratory study by LC-MS/MS

Oxidized LDL (oxLDL) has been shown to play a crucial role in the onset and development of cardiovascular disorders. The study of oxLDL, as an initiator of inflammatory cascades, led to the discovery of a variety of oxidized phospholipids (oxPLs) responsible for pro-inflammatory actions. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) is frequently used by the scientific community as a representative oxPL mixture to study the biological effects of oxidized lipids, due to the high abundance of PAPC in human tissues and the biological activities of oxidized arachidonic acids derivatives. Most studies focusing on oxPAPC effects rely on in-house prepared mixtures of oxidized species obtained by exposing PAPC to air oxidation. Here, we described a multi-laboratory evaluation of the compounds in oxPAPC by LC-MS/MS, focusing on the identification and relative quantification of the lipid peroxidation products (LPPs) formed. PAPC was air-oxidized in four laboratories using the same protocol for 0, 48, and 72 h. It was possible to identify 55 different LPPs with unique elemental composition and characterize different structural isomeric species within these. The study showed good intra-sample reproducibility and similar qualitative patterns of oxidation, as the most abundant LPPs were essentially the same between the four laboratories. However, there were substantial differences in the extent of oxidation, i.e. the amount of LPPs relative to unmodified PAPC, at specific time points. This shows the importance of characterizing air-oxidized PAPC preparations before using them for testing biological effects of oxidized lipids, and may explain some variability of effects reported in the literature

Comprehensive lipidome of human plasma using minimal sample manipulation by liquid chromatography coupled with mass spectrometry

12 Pág. Centro de Biotecnología y Genómica de Plantas (CBGP)[Rationale],The present work shows comprehensive chromatographic methods and MS conditions that have been developed based on the chemical properties of each lipid subclass to detect low-abundance molecular species. This study shows that the developed methods can detect low- and/or very-low-abundant lipids like phosphatidic acid (PA) in the glycerophospholipid (GP) method; dihydroceramide (dhCer) and dihydrosphingosine/sphinganine (dhSPB) in the sphingolipid (SP) method; and lysophosphatidic acid (LPA), LPI, LPG and sphingosine-1-phosphate (SPBP) in the lysolipid method.[Methods], An optimised method for the extraction of lysolipids in plasma is used in addition to Folch extraction. Then, four chromatographic methods coupled with mass spectrometry using targeted and untargeted approaches are described here. Three of the methods use a tertiary pumping system to enable the inclusion of a gradient for analyte separation (pumps A and B) and an isocratic wash (pump C). This wash solution elutes interfering compounds that could cause background signal in the subsequent injections, reducing column lifetime.[Results], Semi-quantitative values for 37 lipid subclasses are reported for a plasma sample (NIST SRM 1950). Furthermore, the methods presented here enabled the identification of 338 different lipid molecular species for GPs (mono- and diacyl-phospholipds), SPs, sterols and glycerolipids. The methods have been validated, and the reproducibility is presented here.[Conclusions], The comprehensive analysis of the lipidome addressed here of glycerolipids, GPs, sterols and SPs is in good agreement with previously reported results, in the NIST SRM 1950 sample, by other laboratories. Ten lipid subclasses LPS, LPI, alkyl-lysophosphatidic acid/alkenyl-lysophosphatidic acid, alkyl-lysophosphatidylethanolamine/alkenyl-lysophosphatidylethanolamine, dhCer (d18:0), SPB (d18:1), dhSPB (d18:0) and SPBP (d18:2) have been detected using this comprehensive method and are uniquely reported here.The authors thank BBSRC for funding to the Babraham Institute. Z.G.K. was funded by the Community of Madrid (FOODAL-CM_S2018/BAAA-4574) and received a grant from the Universidad Politécnica de Madrid and Banco Santander for a Programa Propio Mobility grant to work at the Babraham Institute. The authors greatly acknowledge the encouragement, mentoring and conversations with Michael Wakelam during the development of these methods. He is an inspiration to them to carry his love for lipids and MS.Peer reviewe

Comprehensive lipidome of human plasma using minimal sample manipulation by liquid chromatography coupled with mass spectrometry

Comprehensive analysis of the lipidome is addressed here by analysing lipid subclasses not easily detected by current high-throughput methods. Abundant lipid subclasses in human plasma are chromatographically separated from low abundance lipids prior to detection, avoiding the need for derivatisation. Lipid subclasses from the de novo lipogenesis and sphingolipids pathways are presented in this work. Three chromatographic methods here were implemented using a tertiary pumping system to allow for the inclusion of a gradient for analyte separation using A and B pumps, while an isocratic wash elutes interfering compounds. The isocratic wash enabled elution of lipid subclasses not targeted within the method that would otherwise cause background signal in the subsequent sample injection and reduction in column lifetime. Four chromatographic methods coupled with mass spectrometry using targeted and untargeted approaches to separate high and low abundance lipid subclasses are described here. An optimised method for the extraction of lysolipids is also used in addition to Folch extraction in human plasma

The Effect of a Pex3 Mutation on Hearing and Lipid Content of the Inner Ear

Peroxisome biogenesis disorders (due to PEX gene mutations) are associated with symptoms that range in severity and can lead to early childhood death, but a common feature is hearing impairment. In this study, mice carrying Pex3 mutations were found to show normal auditory development followed by an early-onset progressive increase in auditory response thresholds. The only structural defect detected in the cochlea at four weeks old was the disruption of synapses below inner hair cells. A conditional approach was used to establish that Pex3 expression is required locally within the cochlea for normal hearing, rather than hearing loss being due to systemic effects. A lipidomics analysis of the inner ear revealed a local reduction in plasmalogens in the Pex3 mouse mutants, comparable to the systemic plasmalogen reduction reported in human peroxisome biogenesis disorders. Thus, mice with Pex3 mutations may be a useful tool to understand the physiological basis of peroxisome biogenesis disorders

Targeted delivery of alcohol-containing payloads with antibody-drug conjugates

We herein describe the cell-specific release of alcohol-containing payloads via a sulfatase-sensitive linker in antibody-drug conjugates (ADCs). The linker shows efficient sulfatase-mediated release and high stability in human and mouse plasma. In vitro evaluation demonstrates potent antigen dependent toxicity towards breast cancer cell lines.</p