18286-49-2Relevant articles and documents
Lipid peroxidation generates body odor component trans-2-nonenal covalently bound to protein in vivo
Ishino, Kousuke,Wakita, Chika,Shibata, Takahiro,Toyokuni, Shinya,Machida, Sachiko,Matsuda, Shun,Matsuda, Tomonari,Uchida, Koji
, p. 15302 - 15313 (2010)
trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis- and trans-N∈-3-[(hept-1-enyl)-4-hexylpyridinium] lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis- and trans-HHP-lysine and confirmed that the 2-nonenallysine adducts were indeed formed during the lipid peroxidation- mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand.
HNE Michael adducts to histidine and histidine-containing peptides as biomarkers of lipid-derived carbonyl stress in urines: LC-MS/MS profiling in Zucker obese rats
Orioli, Marica,Aldini, Giancarlo,Benfatto, Maria Carmela,Facino, Roberto Maffei,Carini, Marina
, p. 9174 - 9184 (2007)
A new liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach, based on the precursor ion scanning technique using a triple-stage quadrupole, has been developed to detect free and protein-bound histidine (His) residues modified by reactive carbonyl species (RCS) generated by lipid peroxidation. This approach has been applied to urines from Zucker obese rats, a nondiabetic animal model characterized by obesity and hyperlipidemia, where RCS formation plays a key role in the development of renal and cardiac dysfunction. The immonium ion of His at m/z 110 was used as a specific product ion of His-containing peptides to generate precursor ion spectra, followed by MS 2 acquisitions of each precursor ion of interest for structural characterization. By this approach, three novel adducts, which are excreted in free form only, have been identified, two of them originating from the conjugation of 4-hydroxy-fraras-2-nonenal (HNE) to His, followed by reduction/oxidation of the aldehyde: His-1,4-dihydroxynonane (His-DHN), His-4-hydroxynonanoic acid (His-HNA), and carnosine-HNE, this last recognized in previous in vitro studies as a new potential biomarker of carbonyl stress. No free His-HNE was found in urines, which was detected only in protein hydrolysates. The same LC-MS/MS method, working in multiple reaction monitoring (MRM) mode, has been developed, validated, and applied to quantitatively profile in Zucker urines both conventional (1,4-dihydroxynonane mercapturic acid, DHN-MA) and the newly identified adducts, except His-HNA. The analytes were separated on a C12 reversed-phase column by gradient elution from 100% A (water containing 5 mM nonafluoropentanoic acid) to 80% B (acetonitrile) in 24 min at a flow rate of 0.2 mL/min and analyzed for quantification in MRM mode by applying the following precursor-to-production transitions m/z 322.2 → 164.1 + 130.1 (DHN-MA), m/z 314.7 → 268.2 + 110.1 (His-DHN), m/z 312.2 → 110.1 + 156.0 (His-HNE), m/z 383.1 → 266.2 + 110.1 (CAR-HNE), m/z 319.2 → 301.6 + 156.5 (H-Tyr-His-OH, internal standard). Precision and accuracy data, as well as the lower limits of quantification in urine, were highly satisfactory (from 0.01 nmol/mL for CAR-HNE, His-DHN, His-HNE, to 0.075 nmol/mL for DHN-MA). The method, applied to evaluate for the first time the advanced lipoxidation end products profile in urine from obese Zucker rats, an animal model for the metabolic syndrome, has proved to be suitable and sensitive enough for testing in vivo the carbonyl quenching ability of newly developed RCS sequestering agents.
NADP+-dependent dehydrogenase activity of carbonyl reductase on glutathionylhydroxynonanal as a new pathway for hydroxynonenal detoxification
Moschini, Roberta,Peroni, Eleonora,Rotondo, Rossella,Renzone, Giovanni,Melck, Dominique,Cappiello, Mario,Srebot, Massimo,Napolitano, Elio,Motta, Andrea,Scaloni, Andrea,Mura, Umberto,Del-Corso, Antonella
, p. 66 - 76 (2015)
An NADP+-dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (EC 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (KM 33 μM, kcat 405 min-1), as it is practically inactive toward trans-4-hydroxy-2-nonenal (HNE) and other HNE-adducted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionylnonanoic-δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power.
A method to produce fully characterized ubiquitin covalently modified by 4-hydroxy-nonenal, glyoxal, methylglyoxal, and malondialdehyde
Colzani, Mara,Criscuolo, Angela,Casali, Gaia,Carini, Marina,Aldini, Giancarlo
, p. 328 - 336 (2016)
Reactive carbonyl species (RCS) and the corresponding protein adducts (advanced glycoxidation or lipoxidation end products, i.e. AGEs and ALEs) are now widely studied from different points of view, since they can be considered as biomarkers, pathogenic factors, toxic mediators and drug targets. One of the main limits of the research in this field is the lack of standardized and fully characterized AGEs and ALEs to be used for biological, toxicological, and analytical studies. In this work, we set up a procedure to prepare and fully characterize a set of AGEs and ALEs by incubating ubiquitin - a model protein selected as target for carbonylation - with four different RCS: 4-hydroxy-trans-2-nonenal (HNE), methylglyoxal (MGO), glyoxal (GO), and malondialdehyde (MDA). After 24 h of incubation, the extent of protein carbonylation was estimated using a recently developed quantitative strategy based on high-resolution mass spectrometry. The resulting AGEs and ALEs were fully characterized by both intact protein and bottom-up analyses in terms of: stoichiometry of the total amount of modified protein, elucidation of the structure of the RCS-deriving adducts, and localization of the RCS-modified amino acids. Each RCS exhibited different reactivity toward ubiquitin, as detected by quantifying the extent of protein modification. The order of reactivity was MGO > GO > HNE > MDA. A variety of reaction products was identified and mapped on lysine, arginine, and histidine residues of the protein. In summary, a highly standardized and reproducible method to prepare fully characterized AGEs/ALEs is here presented.
Isotopic labelling for the characterisation of HNE-sequestering agents in plant-based extracts and its application for the identification of anthocyanidins in black rice with giant embryo
Colzani, Mara,Regazzoni, Luca,Criscuolo, Angela,Baron, Giovanna,Carini, Marina,Vistoli, Giulio,Lee, Yoon-Mi,Han, Sang-Ik,Aldini, Giancarlo,Yeum, Kyung-Jin
, p. 896 - 906 (2018)
Reactive carbonyl species (RCS) are cytotoxic molecules that originate from lipid peroxidation and sugar oxidation. Natural derivatives can be an attractive source of potential RCS scavenger. However, the lack of analytical methods to screen and identify bioactive compounds contained in complex matrices has hindered their identification. The sequestering actions of various rice extracts on RCS have been determined using ubiquitin and 4-hydroxy-2-nonenal (HNE) as a protein and RCS model, respectively. Black rice with giant embryo extract was found to be the most effective among various rice varieties. The identification of bioactive compounds was then carried out by an isotopic signature profile method using the characteristic isotopic ion cluster generated by the mixture of HNE: 2H5-HNE mixed at a 1:1 stoichiometric ratio. An in-house database was used to obtain the structures of the possible bioactive components. The identified compounds were further confirmed as HNE sequestering agents through HPLC-UV analysis.
Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE): LC-ESI-MS/MS evidence for Cys374 Michael adduction
Aldini, Giancarlo,Dalle-Donne, Isabella,Vistoli, Giulio,Facino, Roberto Maffei,Carini, Marina
, p. 946 - 954 (2005)
We demonstrate for the first time, by a combined mass spectrometric and computational approach, that G- and F-actin can be covalently modified by the lipid-derived aldehyde, 4-hydroxy-trans-2-nonenal, providing information on the molecular mass of modified protein and the mechanism and site of adduction. ESI-MS analysis of actin treated with different molar ratios of HNE (1:1 to 1:20) showed the formation of a protein derivative in which there was an increase of 156 Da (42028 Da) over native actin (41872 Da), consistent with the adduction of one HNE residue through Michael addition. To identify the site of HNE adduction, G- and F-actin were stabilized by NaBH4 reduction and digested with trypsin. LC-ESI-MS/MS analysis in data-dependent scan mode of the resulting peptides unequivocally indicated that Cys374 is the site of HNE adduction. Computational studies showed that the reactivity of Cys374 residue is due to a significant accessible surface and substantial thiol acidity due to the particular microenvironment surrounding Cys374. Copyright
Detoxification of 4-hydroxynonenal (HNE) in keratinocytes: Characterization of conjugated metabolites by liquid chromatography/electrospray ionization tandem mass spectrometry
Aldini, Giancarlo,Granata, Paola,Orioli, Marica,Santaniello, Enzo,Carini, Marina
, p. 1160 - 1168 (2003)
Keratinocytes are potential targets of lipid peroxidation products (α,β-unsaturated aldehydes) generated in the skin following UV exposure, among which the most abundant and toxic product is 4-hydroxy-trans-2,3-nonenal (HNE). The aim of this study was to investigate the ability of keratinocytes (NCTC2544 cell lines) to detoxify HNE, through characterization of metabolites, until now never demonstrated, using a combined analytical approach (liquid chromatography (LC) and liquid chromatography/mass spectrometry (LC/MS)). Incubation of cells with HNE (up to 200 μM) was performed in order to evaluate the ability of the cells to detoxify this toxic aldehyde, and indicated that the cell viability was maintained under these conditions. LC analysis of the extracellular media from keratinocytes incubated with 100 μM HNE shows a time-dependent decrease of HNE, disappearance from the medium within 2 h and concomitant formation of two unconjugated (phase I) metabolites, 4-hydroxy-2-nonenoic acid (HNA) and 1,4-dihydroxy-2-nonene (DHN), which were both identified and quantified by LC and accounted for 48.8 ± 4.6% of the HNE dose. Four additional metabolites were identified in the extracellular medium by reversed-phase LC coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with positive and negative ion detection as Michael adducts (phase II metabolites), arising by the addition of the nucleophilic sulfur of glutathione (GSH) to the electrophilic C-3 of HNE, followed by oxidation-reduction enzymatic processes. The GSH-HNE conjugates were (a) S-(4-hydroxynonanal-3-yl)glutathione, (b) S-(1,4-dihydroxy-nonane-3-yl)glutathione, (c) S-(4-oxononanal-3-yl)glutathione and (d) S-(4-oxo-nonan-1-ol-3-yl)glutathione, and accounted for 52.3 ± 5.8% of the HNE dose (35 nmol mg-1 protein), as estimated indirectly by measuring the extent of cellular GSH consumption (18.7 ± 1.8 nmol mg-1 protein). The time course of HNE biotransformation was then determined by monitoring the formation of metabolites inside and outside the cell at different times after HNE addition (5-120 min). A time-dependent and almost linear formation inside the cell was observed for all the metabolites (plateau after 15 min of incubation), followed by a rapid decay and a concomitant increase in the extracellular medium (plateau of formation after 60 min). This confirms that HNE diffuses into the cell where is totally metabolized through phase I and phase II reactions to unreactive products, which are then exported outside the cell. This is the first demonstration that skin epidermal cells are able to detoxify the cytotoxic products of lipid peroxidation. Copyright
Formation of 4-hydroxynonenal from cardiolipin oxidation: Intramolecular peroxyl radical addition and decomposition
Liu, Wei,Porter, Ned A.,Schneider, Claus,Brash, Alan R.,Yin, Huiyong
, p. 166 - 178 (2011)
We report herein that oxidation of a mitochondria-specific phospholipid tetralinoleoyl cardiolipin (L4CL) by cytochrome c and H 2O2 leads to the formation of 4-hydroxy-2-nonenal (4-HNE) via a novel chemical mechanism that involves cross-chain peroxyl radical addition and decomposition. As one of the most bioactive lipid electrophiles, 4-HNE possesses diverse biological activities ranging from modulation of multiple signal transduction pathways to the induction of intrinsic apoptosis. However, where and how 4-HNE is formed in vivo are much less understood. Recently a novel chemical mechanism has been proposed that involves intermolecular dimerization of fatty acids by peroxyl bond formation; but the biological relevance of this mechanism is unknown because a majority of the fatty acids are esterified in phospholipids in the cellular membrane. We hypothesize that oxidation of cardiolipins, especially L4CL, may lead to the formation of 4-HNE via this novel mechanism. We employed L4CL and dilinoleoylphosphatidylcholine (DLPC) as model compounds to test this hypothesis. Indeed, in experiments designed to assess the intramolecular mechanism, more 4-HNE is formed from L4CL and DLPC oxidation than 1-palmitoyl-2-linoleoylphosphatydylcholine. The key products and intermediates that are consistent with this proposed mechanism of 4-HNE formation have been identified using liquid chromatography-mass spectrometry. Identical products from cardiolipin oxidation were identified in vivo in rat liver tissue after carbon tetrachloride treatment. Our studies provide the first evidence in vitro and in vivo for the formation 4-HNE from cardiolipin oxidation via cross-chain peroxyl radical addition and decomposition, which may have implications in apoptosis and other biological activities of 4-HNE.
γ-p-toluenesulfonyl-α,β-epoxysilane: A new and practical acrolein β-anion equivalent
Sasaki, Michiko,Takeda, Kei
, p. 4849 - 4851 (2004)
(Chemical Equation Presented) Reaction of γ-p-toluenesulfonyl- α,β-epoxysilane with alkyl halides and aldehydes followed by treatment with n-Bu4NF affords α,β-unsaturated aldehydes via a Brook rearrangement-mediated tandem process under extremely mild conditions.
1,4-dihydroxynonene mercapturic acid, the major end metabolite of exogenous 4-hydroxy-2-nonenal, is a physiological component of rat and human urine
Alary, Jacques,Debrauwer, Laurent,Fernandez, Yvette,Cravedi, Jean-Pierre,Rao, Dinesh,Bories, Georges
, p. 130 - 135 (1998)
In the present study 1,4-dihydroxynonene mercapturic acid (DHN-MA), previously shown to be the major urinary metabolite of 4-hydroxy-2-nonenal (HNE) administered to the rat, was characterized and determined to be a normal constituent of rat and human urine. DHN-MA was excreted as a mixture of at least two stereoisomers as determined by ion trap LC-MS/MS/MS after solid-phase extraction and HPLC purification. The 24-h urinary excretion of this compound was about 10 ng and 5 μg for rat and human, respectively. This end metabolite of the lipid peroxidation product HNE could represent a specific and noninvasive biomarker.
