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

Analysis of SMALP co-extracted phospholipids shows distinct membrane environments for three classes of bacterial membrane protein

Biological characterisation of membrane proteins lags behind that of soluble proteins. This reflects issues with the traditional use of detergents for extraction, as the surrounding lipids are generally lost, with adverse structural and functional consequences. In contrast, styrene maleic acid (SMA) copolymers offer a detergent-free method for biological membrane solubilisation to produce SMA-lipid particles (SMALPs) containing membrane proteins together with their surrounding lipid environment. We report the development of a reverse-phase LC-MS/MS method for bacterial phospholipids and the first comparison of the profiles of SMALP co-extracted phospholipids from three exemplar bacterial membrane proteins with different topographies: FtsA (associated membrane protein), ZipA (single transmembrane helix), and PgpB (integral membrane protein). The data showed that while SMA treatment per se did not preferentially extract specific phospholipids from the membrane, SMALP-extracted ZipA showed an enrichment in phosphatidylethanolamines and depletion in cardiolipins compared to the bulk membrane lipid. Comparison of the phospholipid profiles of the 3 SMALP-extracted proteins revealed distinct lipid compositions for each protein: ZipA and PgpB were similar, but in FtsA samples longer chain phosphatidylglycerols and phosphatidylethanolamines were more abundant. This method offers novel information on the phospholipid interactions of these membrane proteins

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

Identification and relative quantification of tyrosine nitration in a model peptide using two-dimensional infrared spectroscopy

Nitration of tyrosine in proteins and peptides is a post-translational modification that occurs under conditions of oxidative stress. It is implicated in a variety of medical conditions, including neurodegenerative and cardiovascular diseases. However, monitoring tyrosine nitration and understanding its role in modifying biological function remains a major challenge. In this work, we investigate the use of electron-vibration-vibration (EVV) two-dimensional infrared (2DIR) spectroscopy for the study of tyrosine nitration in model peptides. We demonstrate the ability of EVV 2DIR spectroscopy to differentiate between the neutral and deprotonated states of 3-nitrotyrosine, and we characterize their spectral signatures using information obtained from quantum chemistry calculations and simulated EVV 2DIR spectra. To test the sensitivity of the technique, we use mixed-peptide samples containing various levels of tyrosine nitration, and we use mass spectrometry to independently verify the level of nitration. We conclude that EVV 2DIR spectroscopy is able to provide detailed spectroscopic information on peptide side-chain modifications and to detect nitration levels down to 1%. We further propose that lower nitration levels could be detected by introducing a resonant Raman probe step to increase the detection sensitivity of EVV 2DIR spectroscopy. (Graph Presented)

Copper complexes as a source of redox active MRI contrast agents

The study reports an advance in designing copper-based redox sensing MRI contrast agents. Although the data demonstrate that copper(II) complexes are not able to compete with lanthanoids species in terms of contrast, the redox-dependent switch between diamagnetic copper(I) and paramagnetic copper(II) yields a novel redox-sensitive contrast moiety with potential for reversibility

Oxidised LDL-lipids increase beta amyloid production by SH-SY5Y cells through glutathione depletion and lipid raft formation

Elevated total cholesterol in midlife has been associated with increased risk of dementia in later life. We have previously shown that low-density lipoprotein (LDL) is more oxidized in the plasma of dementia patients, although total cholesterol levels are not different from those of age-matched controls. β-Amyloid (Aβ) peptide, which accumulates in Alzheimer disease (AD), arises from the initial cleavage of amyloid precursor protein by β-secretase-1 (BACE1). BACE1 activity is regulated by membrane lipids and raft formation. Given the evidence for altered lipid metabolism in AD, we have investigated a mechanism for enhanced Aβ production by SH-SY5Y neuronal-like cells exposed to oxidized LDL (oxLDL). The viability of SH-SY5Y cells exposed to 4 μg oxLDL and 25 μM 27-hydroxycholesterol (27OH-C) was decreased significantly. Lipids, but not proteins, extracted from oxLDL were more cytotoxic than oxLDL. In parallel, the ratio of reduced glutathione (GSH) to oxidized glutathione was decreased at sublethal concentrations of lipids extracted from native and oxLDL. GSH loss was associated with an increase in acid sphingomyelinase (ASMase) activity and lipid raft formation, which could be inhibited by the ASMase inhibitor desipramine. 27OH-C and total lipids from LDL and oxLDL independently increased Aβ production by SH-SY5Y cells, and Aβ accumulation could be inhibited by desipramine and by N-acetylcysteine. These data suggest a mechanism whereby oxLDL lipids and 27OH-C can drive Aβ production by GSH depletion, ASMase-driven membrane remodeling, and BACE1 activation in neuronal cells. © 2014 The Authors

Genética molecular del virus del síndrome reproductor y respiratorio porcino : aspectos evolutivos, diagnósticos e inmunógenos

El síndrome reproductor y respiratorio porcino esta producido por un virus arn, encuadrado en una reciente familia: arteriviridae. Se han desarrollado tres objetivos en este trabajo: 1/ el desarrollo y puesta a punto de una técnica rt-pcr para el diagnostico directo de la enfermedad; 2/ la expresión de la proteína codificada por la orf-5 vírica tanto en sistemas procariotas como eucariotas; 3/ el estudio de variabilidad existente entre cepas europeas del virus. Los resultados del primer objetivo indican que la técnica puesta a punto es altamente sensible y especifica, permitiendo la detección de aproximadamente 100 di50ct en muestras de campo. Con respecto al segundo objetivo, se ha logrado la caracterización de la antes mencionada proteína, así como la purificación parcial de ella. Por ultimo, los estudios de variabilidad demostraron una elevada semejanza entre las cepas analizadas, aunque si bien mayor entre las de un mismo paí