Dodecenoic Acid Research Articles

Synthesis of 12-aminododecenoic acid by coupling transaminase to oxylipin pathway enzymes

Biobased polymers derived from plant oils are sustainable alternatives to petro based polymers. In recent years, multienzyme cascades have been developed for the synthesis of biobased ω-aminocarboxylic acids, which serve as building blocks for polyamides. In this work, we have developed a novel enzyme cascade for the synthesis of 12-aminododeceneoic acid, a precursor for nylon-12, starting from linoleic acid. Seven bacterial ω-transaminases (ω-TAs) were cloned, expressed in Escherichia coli and successfully purified by affinity chromatography. Activity towards the oxylipin pathway intermediates hexanal and 12-oxododecenoic acid in their 9(Z) and 10(E) isoforms was demonstrated for all seven transaminases in a coupled photometric enzyme assay. The highest specific activities were obtained with ω-TA from Aquitalea denitrificans (TRAD), with 0.62 U mg−1 for 12-oxo-9(Z)-dodecenoic acid, 0.52 U mg−1 for 12-oxo-10(E)-dodecenoic acid and 1.17 U mg−1 for hexanal. A one-pot enzyme cascade was established with TRAD and papaya hydroperoxide lyase (HPLCP-N), reaching conversions of 59% according to LC-ELSD quantification. Starting from linoleic acid, up to 12% conversion to 12-aminododecenoic acid was achieved with a 3-enzyme cascade comprising soybean lipoxygenase (LOX-1), HPLCP-N and TRAD. Higher product concentrations were achieved by the consecutive addition of enzymes compared to simultaneous addition at the beginning.Key points• Seven ω-transaminases converted 12-oxododecenoic acid into its corresponding amine.• A three-enzyme cascade with lipoxygenase, hydroperoxide lyase, and ω-transaminase was established for the first time.• A one-pot transformation of linoleic acid to 12-aminododecenoic acid, a precursor of nylon-12 was achieved.Graphical

GCMS and FTIR analysis of ethanol and methanol leave extract of Urena lobata (Caesar weed) for bioactive phytochemical constituents

This study was designed to apply the highly sophisticated biological and chemical characterization techniques–(GC-MS) Gas chromatography-mass spectrometry and FTIR spectroscopy to screen for bioactive phytocompounds present in ethanol and methanol extracts of U. lobata .The leaves of the plant were collected from a farm in Umuode village in Osisioma Ngwa local government area of Abia state, Nigeria. Ethanol and methanol extracts of the leaves were prepared and analyzed using Buck M910 Gas chromatography system with HP-5MS column (30m in length x250µm in diameter x 0.25µm in thickness film). Analysis of functional groups in the phytocompounds were done using Buck scientific M530 USA FTIR.12 vibrational functional groups were revealed by the FTIR screening in the ethanol and methanol extracts of U. lobata leaves. Some of the functional groups were indicative of alcohols, phenols, aromatic compounds, unsaturated hydrocarbons, vinyl ethers, amines, isonitriles and aliphatic compounds. GC-MS analysis of the ethanol leaf extract revealed 41 bioactive compounds with the following being most abundant, 9-octadecenoic acid(16.8%), dodecenoic acid(13.43%),n-hexadecanoic acid(11.73%), octadecanoic acid (9.78%), 1-docosene(9.57%) while the methanol extract revealed 47 bioactive compounds with the following having the highest abundance, n-hexadecanoic acid (26.65%) and (9.11%), dodecanoic acid(6.89%), 1-docosene(6.06%), erucic acid(4.09%).These phytocompounds and many others present in the leaf have been reported to possess multiple therapeutic activities. This therefore explains the use of this leaf in ancient medicine to treat numerous disease conditions. It further implies that the leaf could be exploited for the formulation of therapeutic molecules.
 Keywords: Urena lobata, GC-MS, FTIR, Spectroscopy, Phytocompounds, Functional group, Phytoconstituents

Synthesis of Polymer Precursor 12-Oxododecenoic Acid Utilizing Recombinant Papaya Hydroperoxide Lyase in an Enzyme Cascade

Hydroperoxide lyases (HPLs) catalyze the splitting of 13S-hydroperoxyoctadecadienoic acid (13S-HPODE) into the green note flavor hexanal and 12-oxo-9(Z)-dodecenoic acid, which is not yet used industrially. Here, HPL from Carica papaya (HPLCP) was cloned and functionally expressed in Escherichia coli to investigate synthesis of 12-oxo-9(Z)-dodecenoic acid in detail. To improve the low catalytic activity of full-length HPLCP, the hydrophobic, non-conserved N-terminal sequence was deleted. This enhanced enzyme activity from initial 10 to 40 U/l. With optimization of solubilization buffer, expression media enzyme activity was increased to 2700 U/l. The tetrameric enzyme was produced in a 1.5 l fermenter and enriched by affinity chromatography. The enzyme preparation possesses a slightly acidic pH optimum and a catalytic efficiency (kcat/KM) of 2.73 × 106 s−1·M−1 towards 13S-HPODE. Interestingly, HPLCP-N could be applied for the synthesis of 12-oxo-9(Z)-dodecenoic acid, and 1 mM of 13S-HPODE was transformed in just 10 s with a yield of 90%. At protein concentrations of 10 mg/ml, the slow formation of the 10(E)-isomer traumatin was observed, pointing to a non-enzymatic isomerization process. Bearing this in mind, a one-pot enzyme cascade starting from safflower oil was developed with consecutive addition of Pseudomonas fluorescens lipase, Glycine max lipoxygenase (LOX-1), and HPLCP-N. A yield of 43% was obtained upon fast extraction of the reaction mixtures after 1 min of HPLCP-N reaction. This work provides first insights into an enzyme cascade synthesis of 12-oxo-9(Z)-dodecenoic acid, which may serve as a bifunctional precursor for bio-based polymer synthesis.

Non-targeted metabolomic analysis predicts the therapeutic effects of exenatide on endothelial injury in patients with type 2 diabetes.

We aimed to investigate whether treatment with exenatide could ameliorate endothelial injury in patients with type 2 diabetes mellitus (T2DM), and to identify biomarkers for predicting amelioration of the endothelial injury induced by the treatment. Ninety-three patients with T2DM were recruited and treated with exenatide for 16 weeks. Enzyme-linked immunosorbent assays were performed at baseline and after the treatment to measure serum levels of endothelial injury markers, including soluble thrombomodulin (sTM). Patients were categorized as responders (n = 47) or non-responders (n = 46) based on median changes in their sTM levels. Serum levels of metabolites at baseline were measured with non-targeted liquid chromatography-mass spectrometry. The results obtained were evaluated with multivariate analysis. Treatment with exenatide for 16 weeks resulted in reduced body weight and improved levels of fasting plasma glucose, 2-hour postprandial plasma glucose, and HbA1c in patients with T2DM (all P < 0.05). Compared with baseline, serum levels of endothelial injury markers including sTM were significantly lowered after the treatment. Metabolites presented at significantly different levels in responders versus non-responders were considered as biomarkers for a therapeutic response of sTM to the exenatide treatment. Among those identified, 4-hydroxyproline and 12-oxo-9(Z)-dodecenoic acid were found to correlate most closely with the exenatide-induced endothelial protection response. The specificity and sensitivity of the multi-metabolite signature model contained higher 4-hydroxyproline and lower 12-oxo-9(Z)-dodecenoic acid were 53.3% and 92.3%, respectively, and the area under receiver operating characteristic curve was 69.2% (P < 0.001). Treatment with exenatide for 16 weeks ameliorates endothelial injury in patients with T2DM. Endothelial protection benefit from exenatide treatment was effectively predicted by the specific metabolomic combination of higher 4-hydroxyproline and lower 12-oxo-9(Z)-dodecenoic acid.

A simple, cost-effective, and rapid separation process for the isolation of anticoagulant active fraction from the fruit extract of Momordica charantia: Characterization of bioactive components and anticoagulant mechanism of active fraction in a mouse model.

A simple, rapid, and cost-effective process for the separation of an active anticoagulant fraction from the aqueous fruit extract of Momordica charantia by using rice husk as adsorbed is described. The in vitro anticoagulant activity of active anticoagulant fraction was comparable to commercial anticoagulants heparin and warfarin. Phytochemical analysis revealed the presence of alkaloids, flavonoids, and phytols in the active anticoagulant fraction, nevertheless; it was devoid of glycosides, triterpenoids, tannins, saponins, steroids, and carbohydrates. By gas chromatography with mass spectrometry analysis, decanoic acid, 1,2,3-propanetriyl ester (22.3%), dodecanoic acid, 1,2,3-propanetriyl ester-d5 (17.3%), dodecenoic acid, 1,2,3-propanetriyl ester (12.5%), and 4-B-methylandrostane 2,3-diol-1,17-dione (11.4%) were identified as the most abundant constituents of active anticoagulant fraction. Presence of αβ-fibrinogenase enzyme was identified by biochemical assay but not by liquid chromatography with tandem mass spectrometry analysis suggesting presence of a novel protease enzyme in this fraction. The active anticoagulant fraction demonstrated biding to fibrinogen but not to thrombin or Factor Xa, inhibited the collagen/ADP-induced mammalian platelet aggregation, showed in vitro thrombolytic activity, noncytotoxic to mammalian cells, showed in vivo plasma defibrinogenation and anticoagulant activities, and inhibited k-carrageen-induced thrombus formation in the tails of mice. Therefore, active anticoagulant fraction (an herbal drug) may find therapeutic application for the prevention and/or treatment of hyperfibrinogenemia/thrombosis-associated cardiovascular disorders.

Identification of characteristic compounds of moderate volatility in breast cancer cell lines.

In this study, we were challenging to identify characteristic compounds in breast cancer cell lines. GC analysis of extracts from the culture media of breast cancer cell lines (MCF-7, SK-BR-3, and YMB-1) using a solid-phase Porapak Q extraction revealed that two compounds of moderate volatility, 1-hexadecanol and 5-(Z)-dodecenoic acid, were detected with markedly higher amount than those in the medium of fibroblast cell line (KMST-6). Furthermore, LC-TOF/MS analysis of the extracts clarified that in addition to the above two fatty acids, the amounts of five unsaturated fatty acids [decenoic acid (C10:1), decadienoic acid (C10:2), 5-(Z)-dodecenoic acid (C12:1), 5-(Z)-tetradecenoic acid (C14:1), and tetradecadienoic acid (C14:2)] in MCF-7 medium were higher than those in medium of KMST-6. Interestingly, H2O2-oxidation of 5-(Z)-dodecenoic acid and 5-(Z)-tetradecenoic acid produced volatile aldehydes that were reported as specific volatiles in breath from various cancer patients, such as heptanal, octanal, nonanal, decanal, 2-(E)-nonenal, and 2-(E)-octenal. Thus, we concluded that these identified compounds over-produced in breast cancer cells in this study could serve as potential precursors producing reported cancer-specific volatiles.

Corrigendum: Progress in and promise of bacterial quorum sensing research.

This corrects the article DOI: 10.1038/nature24624.

Cyclic fatty acids found in frying oils are detoxified via classical drug metabolic pathway but also by β-oxidation and eliminated as conjugates in rats.

Cyclic fatty acid monomers (CFAM) are mainly formed during heat treatments, such as frying, of edible oils. These fatty acids are mixtures of disubstituted five- or six-carbon-membered ring structures. Some earlier studies have suggested that some of these molecules could be metabolized and detoxified, but so far, neither the detoxification mechanisms nor the metabolite identifications have been elucidated. The objective of the present study was to identify the metabolites resulting from the metabolism and detoxification of CFAM. A deuterium-labeled CFAM, [9-(2)H]-10-(6-propyl-2-cyclohexenyl)-dodecenoic acid, was synthesized and fed to rats for 3 days, along with a standard chow diet while the control group was fed the same chow diet which did not contain any CFAM. Biological fluids (urine, blood) were collected for both groups of rats and analyzed using an untargeted metabolomic approach by ultra-performance liquid chromatography coupled with mass spectrometry. Two discriminant metabolites and 18 molecules derived from CFAM were identified or tentatively identified in plasma and urine samples, respectively. The structures of the metabolites suggest that CFAM having a six-carbon-membered ring could be detoxified by the classical drug metabolic pathway (phase I and phase II reactions), but our study also indicates that these are substrates for the β-oxidation pathway and eliminated as glucuronide, sulphate, and/or nitrate conjugates. Urine metabolomics investigations without diet effects have indicated a higher excretion of medium-chain acylcarnitines in the D-CFAM diet group, which may indicate an incomplete β-oxidation.

Abstract 512: Cell-based analysis of oxidative stress, lipid peroxidation and lipid peroxidation-derived protein modifications using fluorescence microscopy

Abstract Oxidative stress plays an important role in the progression of several diseases including inflammation, atherosclerosis, aging and age-related degenerative disorders. Reactive oxygen species damage membrane bound lipids resulting in lipid peroxidation-derived protein modifications. Cell-based measurements of oxidative stress, lipid peroxidation and protein carbonylation by traditional fluorescence microscopy provide a powerful platform to quantitate oxidative stress and lipid peroxidation. Here, we used three different approaches to measure oxidative stress and lipid peroxidation in cells by fluorescence microscopy. 1) Two new fluorogenic probes, CellROX™ Orange and CellROX ™ Green Reagents to measure oxidative stress in cells, 2) Image-iT® Lipid Peroxidation Kit for a ratiometric determination of lipid peroxidation in live cells 3) Click-iT® Lipid Peroxidation Imaging Kit, a click chemistry-based approach which utilizes incorporation of an alkyne-modified unsaturated fatty acid analog, linoleamide, into the cellular membranes. The resulting oxidation products, like 9, 12-dioxo-10(E) dodecenoic acid (DODE) can readily modify proteins and these modifications were readily detected in fixed cells by the copper-catalyzed click reaction using fluorescent azides. Using these approaches, we measured oxidative stress and lipid peroxidation caused by several oxidants in cells. Increases in oxidative stress, lipid peroxidation, and protein modifications were assessed by high content imaging and analysis as well as traditional fluorescence microscopy. In the models tested, at least 2-3 fold increases were observed compared to controls was and responses were successfully inhibited by antioxidants. The three strategies described here provide powerful new tools for the assessment of oxidative stress in cells and convey distinct advantages over existing cell-based methods. Citation Format: Bhaskar S. Mandavilli, Robert Aggeler. Cell-based analysis of oxidative stress, lipid peroxidation and lipid peroxidation-derived protein modifications using fluorescence microscopy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 512. doi:10.1158/1538-7445.AM2014-512

Constituents of the essential oil and fatty acid from Malania oleifera

The essential oil and fatty acid composition of Malania oleifera were determined by GC–MS techniques. The yield of essential oils was 0.1±0.02%, 0.2±0.03% and 1.1±0.12%, respectively from seeds, branches with leaves and outer part of pericarp (exocarp and mesocarp) of M. oleifera. Seventeen compounds were identified in the essential oils of seeds with benzyl alcohol (58.42%), benzaldehyde (29.66%) and benzoic acid (1.49%) as main constituents. Both branches with leaves and outer part of pericarp, the major constituents include benzaldehyde (87.15% and 90.68%), benzyl alcohol (6.51% and 2.95%), benzeneacetonitrile (5.66% and 0.07%) as the most abundant components. These results indicated that benzaldehyde and benzyl alcohol were found as the main essential oils obtained from the different parts of M. oleifera. The yield of fatty acids extracted from the seeds was 60.5±2.5%, which contains fourteen chromatographic components, of which 10 compounds were identified, representing 98.98% of the lipids; the major components were nervonic acid (55.70%), octadecenoic acid (23.81%), docosenoic acid (13.13%), tetracosanoic acid (2.65%), dodecenoic acid (1.28%), tricosadienoic acid (0.99%) and hexadecanoic acid (0.45%). The results demonstrated that the quantities of unsaturated fatty acids were higher than that of saturated analog.

Fragmentation of a linoleate-derived γ-hydroperoxy-α,β-unsaturated epoxide to γ-hydroxy- and γ-oxo-alkenals involves a unique pseudo-symmetrical diepoxycarbinyl radical

Many of the pathological effects of lipid peroxidation are mediated by aldehydes generated through fragmentation of lipid peroxides. Among these aldehydes, the γ-hydroxy- and γ-oxo-α,β-alkenals, e.g., 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), are especially prone to modifying proteins and DNA through covalent adduction. In addition the “mirror image” γ-hydroxy- and γ-oxo-α,β-alkenal phospholipids can serve as high-affinity ligands for biological receptors triggering pathology. Therefore, the mechanisms by which these aldehydes are generated in vivo are under intense scrutiny. We now report observations supporting the intermediacy of a unique pseudo-symmetrical diepoxycarbinyl radical that accounts for the coproduction of HNE, ONE, and their mirror image analogues 9-hydroxy-12-oxo-10(E)-dodecenoic acid and 9-keto-12-oxo-10-dodecenoic acid upon fragmentation of 13-hydroperoxy-cis-9,10-epoxyoctadeca-11-enoic acid.

C12 derivatives of the hydroperoxide lyase pathway are produced by product recycling through lipoxygenase‐2 in Nicotiana attenuata leaves

In response to diverse stresses, the hydroperoxide lyase (HPL) pathway produces C(6) aldehydes and 12-oxo-(9Z )-dodecenoic acid ((9Z )-traumatin). Since the original characterization of (10E )-traumatin and traumatic acid, little has been added to our knowledge of the metabolism and fluxes associated with the conversion of (9Z )-traumatin into diverse products in response to wounding and herbivory. A liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method was developed to quantify C(12) derivatives of the HPL pathway and to determine their metabolism after wounding and simulated herbivory in Nicotiana attenuata leaves. Ninety-eight per cent of the (9Z )-traumatin produced was converted to 9-hydroxy-(10E )-traumatin (9-OH-traumatin); two-thirds by product recycling through lipoxygenase-2 (NaLOX2) activity and one-third by nonenzymatic oxidation. Thirty-eight per cent of the de novo produced 9-OH-traumatin was conjugated to glutathione, consistent with this oxylipin being a reactive electrophile species. 12-OH-(9Z )-dodecenoic and dodecenedioic acids also showed rapid increases after wounding and simulated herbivory and a role for C(12) derivatives as signals in these processes was consistent with their ability to elicit substantial changes in gene expression. These results underscore the importance of metabolite reflux through LOX2, an insight which creates new opportunities for a functional understanding of C(12) derivatives of the HPL pathway in the regulation of stress responses.

Proteins Modified by the Lipid Peroxidation Aldehyde 9,12-Dioxo-10(E)-dodecenoic Acid in MCF7 Breast Cancer Cells

The hydroperoxide of linoleic acid (13-HPODE) degrades to 9,12-dioxo-10(E)-dodecenoic acid (DODE), which readily modifies proteins. This study identified the major proteins in MCF7 cells modified by DODE. To reduce false positives, three methods were used to identify DODE-modified proteins. First, cells were treated with a synthetically biotinylated 13-HPODE (13-HPODE-biotin). Modified proteins were enriched by neutravidin affinity and identified by two-dimensional liquid chromatography--tandem mass spectrometry (2D LC-MS/MS). Second, cells were treated with native 13-HPODE. Protein carbonyls were biotinylated with an aldehyde reactive probe, and modified proteins were enriched by neutravidin affinity and identified by 2D LC-MS/MS. Third, using a newly developed DODE antibody, DODE-modified proteins were located by 2D sodium dodecyl sulfate--polyacrylamide gel electrophoresis and Western blot and identified by in-gel digestion and LC-MS/MS. Analysis of the proteins characterized by all three methods revealed a significant overlap and identified 32 primary proteins modified by DODE in MCF7 cells. These results demonstrated the feasibility for the cellular formation of DODE protein-carbonyl adducts that may be future indicators of oxidative stress.

Mass Spectrometric Characterization of Protein Modification by the Products of Nonenzymatic Oxidation of Linoleic Acid

Autoxidation of linoleic acid (LA) enhanced by Fe(II)/ascorbate generates unsaturated hydroperoxides which undergo further oxidative evolution resulting in a mixture of electrophiles, including epoxyketooctadecenoic acid and dienones with intact C-18 chains as well as oxidative cleavage products such as 4-hydroxy-2(E)-nonenal (HNE), 4-oxo-2(E)-nonenal (ONE), 2(E)-octenal, 9-hydroxy-12-oxo-10(E)-dodecenoic acid, 9,12-dioxo-10(E)-dodecenoic acid, and 11-oxoundec-9(E)-enoic acid. Mass spectrometric (MALDI-TOF-MS and LC-ESI-MS/MS) studies have been performed following incubation of the model protein beta-lactoglobulin with LA, Fe(II), and ascorbate, which identified adducts of these electrophiles with three different protein nucleophiles. Deuterium labeled linoleic acid 17,17,18,18,18-d(5)-(9Z,12Z)-octadeca-9,12-dienoic acid (d(5)-LA) was synthesized to facilitate the detection and characterization of the protein modifications by mass spectrometry. Reduction by NaBH(4) served to trap reversible adducts and to quantify the number of reducible functional groups in each adduct. This study, which mimics the distribution of reactive lipid peroxidation products generated by a continuous low level flux of reactive oxygen species present in vivo under conditions of oxidative stress, confirms that many irreversibly formed adducts previously identified following exposure of model proteins to pure electrophilic modifiers such as HNE and ONE are also generated during in situ oxidation of LA. These adducts include HNE-His Michael adducts (MA), ONE-Lys 4-ketoamide, ONE-Lys pyrrolinone, and a Cys/His-ONE-Lys pyrrole cross-link. However, reversibly formed adducts, such as the HNE-Lys Schiff base, are not present at detectable levels. The isotopic labeling allowed less commonly identified mirror-image adducts derived from the carboxy terminus of LA to be identified. A novel 2-octenoic acid-His MA was discovered.

Nitro-fatty Acid Metabolome: Saturation, Desaturation, β-Oxidation, and Protein Adduction

Nitrated derivatives of fatty acids (NO2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO2-FA signal transduction lacks insight into how or if NO2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO2) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO2 or metabolites were detected under basal conditions, whereas administered 18:1-NO2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO2-FA are also metabolized via beta-oxidation, with high performance liquid chromatography-tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO2 as metabolites. Additionally, a significant proportion of 18:1-NO2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO2 hydration or nitric oxide (*NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO2 detected by exchange to added beta-mercaptoethanol. This, coupled with the observation of 18:1-NO2 release from glutathione-18:1-NO2 adducts, supports that reversible and exchangeable NO2-FA-thiol adducts occur under biological conditions. After administration of [3H]18:1-NO2, 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and beta-oxidation reactions that terminate at the site of acyl-chain nitration.

Identification of Proteins Adducted by Lipid Peroxidation Products in Plasma and Modifications of Apolipoprotein A1 with a Novel Biotinylated Phospholipid Probe

Reactive electrophiles generated by lipid peroxidation are thought to contribute to cardiovascular disease and other oxidative stress-related pathologies by covalently modifying proteins and affecting critical protein functions. The difficulty of capturing and analyzing the relatively small fraction of modified proteins complicates identification of the protein targets of lipid electrophiles. We recently synthesized a biotin-modified linoleoylglycerylphosphatidycholine probe called PLPBSO (Tallmanet al.Chem. Res. Toxicol.2007, 7, 227−23417305406), which forms typical linoleate oxidation products and covalent adducts with model peptides and proteins. Supplementation of human plasma with PLPBSO followed by free radical oxidation resulted in covalent adduction of PLPBSO to plasma proteins, which were isolated with streptavidin and identified by liquid chromatography-tandem mass spectrometry (LC-MS−MS). Among the most highly modified proteins was apolipoprotein A1 (ApoA1), which is the core component of high density lipoprotein (HDL). ApoA1 phospholipid adduct sites were mapped by LC-MS−MS of tryptic peptides following mild base hydrolysis to release esterified phospholipid adducts. Several carboxylated adducts formed from phospholipid-esterified 9,12-dioxo-10(E)-dodecenoic acid (KODA), 9-hydroxy, 12-oxo-10(E)-dodecenoic acid (HODA), 7-oxoheptanoic acid, 8-oxooctanoic acid, and 9-oxononanoic acid were identified. Free radical oxidations of isolated HDL also generated adducts with 4-hydroxynonenal (HNE) and other noncarboxylated electrophiles, but these were only sporadically identified in the PLPBSO-adducted ApoA1, suggesting a low stoichiometry of modification in the phospholipid-adducted protein. Both phospholipid electrophiles and HNE adducted His162, which resides in an ApoA1 domain involved in the activation of Lecithin-cholesterol acyltransferase and maturation of the HDL particle. ApoA1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress.

Growth inhibition of the eight species of microalgae by growth inhibitor from the culture of Isochrysis galbana and its isolation and identification

A type of growth inhibitor was successfully isolated and purified from cell-free filtrates of cultural medium at the death phase of Isochrysis galbana, and its chemical structure was confirmed by the methods of FABMS, UV, 1H-NMR, 13C-NMR and 2D NMR, which was 1-[hydroxyl-diethyl malonate]-isopropyl dodecenoic acid, C22H38O7. The results showed that the growth-inhibitor strongly inhibited the growth of Isochrysis galbana, and the growth of the eight species of microalgae (Dunaliella salina, Platymonas elliptica, Chlorella vugralis, Nitzschia closterium, Chaetoceros muelleri, Chaetoceros gracilis, Nitzschia closterium minutissima, Phaeodactylum tricornutum) also could be regulated by the growth-inhibitor in a concentration-dependent manner. The further investigation found that the synthesis process of chlorophyll and protein in the cells of all test microalgae could be inhibited by the growth inhibitor, and the content of chlorophyll and protein significantly decreased.

Covalent Adducts Arising from the Decomposition Products of Lipid Hydroperoxides in the Presence of Cytochrome c

Polyunsaturated fatty acids can be converted to lipid hydroperoxides through nonenzymatic and enzymatic pathways. The prototypic omega-6 lipid hydroperoxide 13-hydroperoxy-octadecadienoic acid (13-HPODE) homolytically decomposes to form highly reactive alpha,beta-unsaturated aldehydes, such as 9,12-dioxo-10(E)-dodecenoic acid (DODE), 4-oxo-2(E)-nonenal (ONE), 4,5-epoxy-2(E)-decenal (EDE), and 4-hydroxy-2(E)-nonenal (HNE), that can form covalent adducts with DNA. Both 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal can also modify proteins to form products that can potentially serve as biomarkers of lipid hydroperoxide-mediated macromolecule damage. In this study, cytochrome c was used to identify and individually characterize the modification sites for each of these aldehydes and also determine the most abundant adduct formed following the decomposition of 13-HPODE. The adducts were characterized by ESI-TOF/MS analysis of the intact proteins and by a combination of ESI-ion-trap/MSn and quadrupole-TOF/MS/MS analysis of the tryptic and chymotryptic peptides. The major adducts included an HNE-His Michael adduct on H33, EDE-Lys adducts on K7 and K8, ONE-Lys ketoamide adducts on K5, K7, and K8, an apparent ONE-Lys Michael adduct on K5, and DODE-Lys carboxyl ketoamide adducts on K86 and K87. DODE was the most reactive aldehyde toward cytochrome c. The major adduct from this reaction was analogous to the most abundant adduct resulting from the decomposition of 13-HPODE in the presence of cytochrome c.

Induction of endothelial cell apoptosis by lipid hydroperoxide-derived bifunctional electrophiles

Endothelial dysfunction is considered to be the earliest event in atherogenesis. Oxidative stress, inflammation, and apoptosis play critical roles in its progression and onset. Lipid peroxidation, which occurs during oxidative stress, results in the formation of lipid hydroperoxide-derived bifunctional electrophiles such as 4-hydroxy-2( E)-nonenal that induce apoptosis. In this study, recently identified lipid hydroperoxide-derived bifunctional electrophiles 4-oxo-2( E)-nonenal (ONE; 5–30 μM) and 4,5-epoxy-2( E)-decenal (EDE; 10–20 μM) were shown to cause a dose- and time-dependent apoptosis in EA.hy 926 endothelial cells. This was manifest by morphological changes, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. Bifunctional electrophiles caused cytochrome c release from mitochondria into the cytosol, implicating a mitochondrial pathway of apoptosis in the endothelial cells. The novel carboxylate-containing lipid hydroperoxide-derived bifunctional electrophile 9,12-dioxo-10( E)-dodecenoic acid was inactive because it could not translocate across the plasma membrane. However, its less polar methyl ester derivative (2–10 μM) was the most potent inducer of apoptosis of any bifunctional electrophile that has been tested. An acute decrease in intracellular glutathione (GSH) preceded the onset of apoptosis in bifunctional electrophile-treated cells. The ability of ONE and EDE to deplete GSH was directly correlated with their predicted reactivity toward nucleophilic amino acids. Liquid chromatography/mass spectrometry methodology was developed in order to examine the intracellular and extracellular concentrations of bifunctional electrophile-derived GSH adducts. Relative intracellular/extracellular ratios of the GSH adducts were identical with the rank order of potency for inducing caspase 3 activation. This suggests that there may be a role for the bifunctional electrophile-derived GSH adducts in the apoptotic response. N-Acetylcysteine rescued bifunctional electrophile-treated cells from apoptosis, whereas the GSH biosynthesis inhibitor d, l-buthionine-( R, S)-sulfoximine sensitized the cells to apoptosis. These data suggest that lipid hydroperoxide-derived bifunctional electrophiles may play an important role in cardiovascular pathology through their ability to induce endothelial cell apoptosis.

Dioxododecenoic Acid: A Lipid Hydroperoxide-Derived Bifunctional Electrophile Responsible for Etheno DNA Adduct Formation

It has been proposed that 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid [13(S)-HPODE]-mediated formation of 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal arises from a Hock rearrangement. This suggested that a 4-oxo-2(E)-nonenal-related molecule, 9,12-dioxo-10(E)-dodecenoic acid (DODE), could also result from the intermediate formation of 9-hydroperoxy-12-oxo-10(E)-dodecenoic acid. A recent report has described the formation of DODE-derived etheno adducts when 13(S)-HPODE was allowed to decompose in the presence of 2'-deoxynucleosides or DNA. However, the regioselectivity of lipid hydroperoxide-derived DODE addition to 2'-deoxyguanosine (dGuo) or other 2'-deoxynucleosides was not determined. The structure of carboxynonanone-etheno-dGuo formed from vitamin C-mediated 13(S)-HPODE decomposition has now been established by a combination of 1H and 13C NMR spectroscopy studies of its bis-methylated derivative. The site of dGuo methylation was first established as being at N-5 rather than at O-9 from NMR analysis of a methyl derivative of the model compound, heptanone-etheno-dGuo. (1)H,(13)C 2D heteronuclear multiple bond correlations were then used to establish unequivocally that the bis-methyl derivative of carboxynonanone-etheno-dGuo was 3-(2'-deoxy-beta-d-erythropentafuranosyl)imidazo-7-(9' '-carboxymethylnona-2' '-one)-9-oxo-5-N-methyl[1,2-a]purine rather than its 6-(9' '-carboxymethylnona-2"-one)-9-oxo-5-N-methyl[1,2-a]purine regioisomer. Therefore, etheno adduct formation occurred by initial nucleophilic attack of the exocyclic N(2) amino group of dGuo at the C-12 aldehyde of DODE to form an unstable carbinolamine intermediate. This was followed by intramolecular Michael addition of the pyrimidine N1 of dGuo to C-11 of the resulting alpha,beta-unsaturated ketone. Subsequent dehydration gave 3-(2'-deoxy-beta-d-erythropentafuranosyl)imidazo-7-(9' '-carboxynona-2' '-one)-9-oxo-[1,2-a]purine (carboxynonanone-etheno-dGuo). An efficient synthesis of DODE was developed starting from readily available 1,8-octanediol using a furan homologation procedure. This synthetic method allowed multigram quantities of DODE to be readily prepared. Synthetic DODE when reacted with dGuo gave carboxynonanone-etheno-dGuo that was identical with that derived from vitamin C-mediated 13(S)-HPODE decomposition in the presence of dGuo.