Activation of stress signaling pathways by electrophilic oxidized and nitrated lipids

Unsaturated fatty acids are prone to radical reactions that occur in biological situations where extensive formation of reactive oxygen and nitrogen species (ROS and RNS) takes place. These reactions are frequent in inflammatory conditions such as atherosclerosis, and yield a variety of biologically active species, many of which are electrophilic in nature. Electrophilic lipid oxidation and nitration products can influence redox cell signaling via S-alkylation of protein thiols, and moderate exposure to these species evokes protective cell signaling responses through this mechanism. Herein, we review the stress signaling pathways elicited by electrophiles derived from unsaturated fatty acids, focusing on the Keap1-Nrf2 pathway, the heat shock response pathway (HSR), and the unfolded protein response pathway (UPR).     

Reactions of *NO, *NO2 and peroxynitrite in membranes: physiological implications

Nitric oxide (^·NO) and nitrogen dioxide (^·NO₂) are hydrophobic gases. Therefore, lipid membranes and hydrophobic regions of proteins are potential sinks for these species. In these hydrophobic environments, reactive nitrogen species will exhibit different chemistry than in aqueous environments due to higher local concentrations and the lack of hydrolysis reactions. The peroxynitrite anion (ONOO^-) and peroxynitrous acid (ONOOH) can freely pass through lipid membranes, making peroxynitrite-mediated reactions in a hydrophobic environment also of extreme relevance. The reactions observed by these reactive nitrogen species in a hydrophobic milieu include oxidation, nitration and even potent chain-breaking antioxidant reactions. The physiological and toxicological relevance of these reactions is discussed.     

Common themes in glycoconjugate assembly using the biogenesis of O-antigen lipopolysaccharide as a model system

The biosynthesis of glycoconjugates is remarkably conserved in all types of cells since the biochemical reactions involved exhibit similar characteristics, which can be summarized as follows: (a) the saccharide moiety is formed as a lipidlinked, membrane-associated glycan; (b) the lipid component in most cases is a polyisoprenoid phosphate; (c) the assembly of the lipid-linked saccharide intermediate depends on reactions taking place at both sides of the cell membrane, which requires the obligatory transmembrane movement of amphipathic molecules across the lipid bilayer. These general characteristics are present in the biosynthesis of the O-antigen component of the bacterial lipopolysaccharide, which serves as a model system to investigate the molecular and mechanistic basis of glycoconjugate synthesis, as summarized in this minireview.     

Nitric oxide reaction with lipid peroxyl radicals spares alpha-tocopherol during lipid peroxidation. Greater oxidant protection from the pair nitric oxide/alpha-tocopherol than alpha-tocopherol/ascorbate

The reactions of nitric oxide ((.)NO) and alpha-tocopherol (alpha-TH) during membrane lipid oxidation were examined and compared with the pair alpha-TH/ascorbate. Nitric oxide serves as a more potent inhibitor of lipid peroxidation propagation reactions than alpha-TH and protects alpha-TH from oxidation. Mass spectrometry, oxygen and (.)NO consumption, conjugated diene analyses, and alpha-TH fluorescence determinations all demonstrated that (.)NO preferentially reacts with lipid radical species, with alpha-TH consumption not occurring until (.)NO concentrations fell below a critical level. In addition, alpha-TH and (.)NO cooperatively inhibit lipid peroxidation, exhibiting greater antioxidant capacity than the pair alpha-TH/ascorbate. Pulse radiolysis analysis showed no direct reaction between (.)NO and alpha-tocopheroxyl radical (alpha-T(.)), inferring that peroxyl radical termination reactions are the principal lipid-protective mechanism mediated by (.)NO. These observations support the concept that (.)NO is a potent chain breaking antioxidant toward peroxidizing lipids, due to facile radical-radical termination reactions with lipid radical species, thus preventing alpha-TH loss. The reduction of alpha-T(.) by ascorbate was a comparatively less efficient mechanism for preserving alpha-TH than (.)NO-mediated termination of peroxyl radicals, due to slower reaction kinetics and limited transfer of reducing equivalents from the aqueous phase. Thus, the high lipid/water partition coefficient of (.)NO, its capacity to diffuse and concentrate in lipophilic milieu, and a potent reactivity toward lipid radical species reveal how (.)NO can play a critical role in regulating membrane and lipoprotein lipid oxidation reactions.     

Superoxide-dependent lipid peroxidation. Problems with the use of catalase as a specific probe for fenton-derived hydroxyl radicals

Hydroxyl radicals (OH.) can initiate lipid oxidation by hydrogen abstraction. Transition metals however, particularly iron and copper, stimulate lipid oxidation by reacting with lipid peroxides to form new radical species. The haem-iron protein catalase can react non-specifically with lipid peroxides in this way resulting in loss of their conjugated diene structures. When a superoxide-generating system is used to stimulate lipid autoxidation, catalase can conceivably inhibit the reaction in two ways (A) by decomposing lipid peroxides as they are formed (B) through the removal of hydrogen peroxide preventing OH. radical formation. Results presented here suggest that the latter interpretation, although commonly presented, cannot be automatically assumed.     

Hydrogen peroxide-mediated inactivation of microsomal cytochrome P450 during monooxygenase reactions

Cytochrome P450 can undergo inactivation following monooxygenase reactions in liver microsomes of untreated, phenobarbital and 3-methylcholanthrene-treated rats and rabbits. The acceleration of cytochrome P450 loss in the presence of catalase inhibitors (sodium azide, hydroxylamine) indicates that hydrogen peroxide is involved in hemoprotein degradation. It was revealed that cytochrome P450 is inactivated mainly by H₂O₂ formed through peroxy complex breakdown, whereas H₂O₂ formed via the dismutation of superoxide anions produces a slight inactivating effect. The hydrogen peroxide added outside or formed by a glucose-glucose oxidase system has less of an inactivating effect than H₂O₂ produced within the cytochrome P450 active center. Self-inactivation of cytochrome P450 during oxygenase reactions is highly specific. Other components of the monooxygenase system, such as cytochrome b₅, NADH- and NADPH-specific flavorproteins, undergo no inactivation. The alterations in phospholipid content and in the rate of lipid peroxidation were not observed as well. The inactivation of cytochrome P450 by H₂O₂ is the result of heme loss or destruction without cytochrome P420 formation. Such. a mechanism operates with different substrates and cytochrome P450 species catalyzing the partially coupled monooxygenase reactions.     

Dehydroepiandrosterone metabolism in human plasma.

The possibility that dehydroepiandrosterone (DHEA) is metabolized in human plasma was studied by column and thin-layer chromatography. The results obtained indicate that a time-dependent disappearance of DHEA is matched by the appearance of newly-formed species that may represent DHEA conversion by-products. Neither disappearance of DHEA, nor formation of the alleged conversion by-products was observed when reactions were performed under conditions in which plasma enzymes were removed or inactivated. These results suggest that, in plasma, DHEA is partially transformed into different substances, and that the conversion reactions are catalyzed by enzymes present in this tissue. The observed kinetics of appearance and partial disappearance of the radiolabeled species can be interpreted as indicating that some of the by-products formed are further converted into other substances. The data shown appear to indicate that plasma can be added to the list of the already known compartments that are involved in steroid metabolism.     

Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury

Increasing appreciation of the causative role of oxidative injury in many disease states places great importance on the reliable assessment of lipid peroxidation. Malondialdehyde (MDA) is one of several low-molecular-weight end products formed via the decomposition of certain primary and secondary lipid peroxidation products. At low pH and elavated temperature, MDA readily participates in nucleophilic addition reaction with 2-thiobarbituric acid (TBA), generating a red, fluorescent 1:2 MDA:TBA adduct. These facts, along with the availability of facile and sensitive methods to quantify MDA (as the free aldehyde or its TBA derivative), have led to the routine use of MDA determination and, particularly, the “TBA test” to detect and quantify lipid peroxidation in a wide array of sample types. However, MDA itself participates in reactions with molecules other than TBA and is a catabolic substrate. Only certain lipid peroxidation products generate MDA (invariably with low yields), and MDA is neither the sole end product of fatty peroxide formation and decomposition nor a substance generated exclusively through lipid peroxidation. Many factors (e.g., stimulus for and conditions of peroxidation) modulate MDA formation from lipid. Additional factors (e.g., TBA-test reagents and constituents) have profound effects on test response to fatty peroxide-derived MDA. The TBA test is intrinsically nonspecific for MDA: nonlipid-related materials as well as fatty peroxide-derived decomposition products other than MDA are TBA positive. These and other considerations from the extensive literature on MDA, TBA reactivity, and oxidative lipid degradation support the conclusion that MDA determination and the TBA test can offer, at best, a narrow and somewhat empirical window on the complex process of lipid peroxidation. The MDA content and/or TBA reactivity of a system provides no information on the precise structures of the “MDA precursor(s),” their molecular origins, or the amount of each formed. Consequently, neither MDA determination nor TBA-test response can generally be regarded as a diagnostic index of the occurrence/extent of lipid peroxidation, fatty hydroperoxide formation, or oxidative injury to tissue lipid without independent chemical evidence of the analyte being measured and its source. In some cases, MDA/TBA reactivity is an indicator of lipid peroxidation; in other situations, no qualitative or quantitative relationship exists among sample MDA content, TBA reactivity, and fatty peroxide tone. Utilization of MDA analysis and/or the TBA test and interpretation of sample MDA content and TBA test response in studies of lipid peroxidation require caution, discretion, and (especially in biological systems) correlative data from other indices of fatty peroxide formation and decomposition.     

Glycation and glycoxidation of low-density lipoproteins by glucose and low-molecular mass aldehydes. Formation of modified and oxidized particles.

Patients with diabetes mellitus suffer from an increased incidence of complications including cardiovascular disease and cataracts; the mechanisms responsible for this are not fully understood. One characteristic of such complications is an accumulation of advanced glycation end-products formed by the adduction of glucose or species derived from glucose, such as low-molecular mass aldehydes, to proteins. These reactions can be nonoxidative (glycation) or oxidative (glycoxidation) and result in the conversion of low-density lipoproteins (LDL) to a form that is recognized by the scavenger receptors of macrophages. This results in the accumulation of cholesterol and cholesteryl esters within macrophages and the formation of foam cells, a hallmark of atherosclerosis. The nature of the LDL modifications required for cellular recognition and unregulated uptake are poorly understood. We have therefore examined the nature, time course, and extent of LDL modifications induced by glucose and two aldehydes, methylglyoxal and glycolaldehyde. It has been shown that these agents modify Arg, Lys and Trp residues of the apoB protein of LDL, with the extent of modification induced by the two aldehydes being more rapid than with glucose. These processes are rapid and unaffected by low concentrations of copper ions. In contrast, lipid and protein oxidation are slow processes and occur to a limited extent in the absence of added copper ions. No evidence was obtained for the stimulation of lipid or protein oxidation by glucose or methylglyoxal in the presence of copper ions, whereas glycolaldehyde stimulated such reactions to a modest extent. These results suggest that the earliest significant events in this system are metal ion-independent glycation (modification) of the protein component of LDL, whilst oxidative events (glycoxidation or direct oxidation of lipid or proteins) only occur to any significant extent at later time points. This 'carbonyl-stress' may facilitate the formation of foam cells and the vascular complications of diabetes.     

A predicted plastid rhomboid protease affects phosphatidic acid metabolism in Arabidopsis thaliana

The thylakoid membranes of the chloroplast harbor the photosynthetic machinery that converts light into chemical energy. Chloroplast membranes are unique in their lipid makeup, which is dominated by the galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG). The most abundant galactolipid, MGDG, is assembled through both plastid and endoplasmic reticulum (ER) pathways in Arabidopsis, resulting in distinguishable molecular lipid species. Phosphatidic acid (PA) is the first glycerolipid formed by the plastid galactolipid biosynthetic pathway. It is converted to substrate diacylglycerol (DAG) for MGDG Synthase (MGD1) which adds to it a galactose from UDP‐Gal. The enzymatic reactions yielding these galactolipids have been well established. However, auxiliary or regulatory factors are largely unknown. We identified a predicted rhomboid‐like protease 10 (RBL10), located in plastids of Arabidopsis thaliana, that affects galactolipid biosynthesis likely through intramembrane proteolysis. Plants with T‐DNA disruptions in RBL10 have greatly decreased 16:3 (acyl carbons:double bonds) and increased 18:3 acyl chain abundance in MGDG of leaves. Additionally, rbl10‐1 mutants show reduced [¹⁴C]–acetate incorporation into MGDG during pulse?chase labeling, indicating a reduced flux through the plastid galactolipid biosynthesis pathway. While plastid MGDG biosynthesis is blocked in rbl10‐1 mutants, they are capable of synthesizing PA, as well as producing normal amounts of MGDG by compensating with ER‐derived lipid precursors. These findings link this predicted protease to the utilization of PA for plastid galactolipid biosynthesis potentially revealing a regulatory mechanism in chloroplasts.     

Docosahexaenoic acid-induced changes in phospholipids in cortex of young and aged rats: a lipidomic analysis

The age-related decline in cognitive function has been associated with biochemical changes that can be attenuated following n-3 polyunsaturated fatty acid treatment. Dietary supplementation with docosahexaenoic acid (DHA) has been shown to reverse age-related changes in synaptic function. Here, lipidomic analyses were undertaken to examine changes in lipid classes and phospholipid species in cortical tissue of young (2-4 months) and aged (20-22 months), control- and DHA-treated (10mg daily) rats following treatment for 8 weeks, aiming to explore the mechanism of DHA action. Dietary supplementation normalised the age-related decrease in unsaturation index, reduced the levels of arachidonic acid-containing phospholipids in both young and aged animals, and gave rise to production of new phosphatidylserine and phosphatidylinositol species. These findings suggest that DHA may mediate some of its effects through alterations in the membrane lipid composition that can consequently affect the production of pro-inflammatory mediators and signalling molecular species.     

Oxidative risk for atherothrombotic cardiovascular disease

In the vasculature, reactive oxidant species, including reactive oxygen, nitrogen, or halogenating species, and thiyl, tyrosyl, or protein radicals may oxidatively modify lipids and proteins with deleterious consequences for vascular function. These biologically active free radical and nonradical species may be produced by increased activation of oxidant-generating sources and/or decreased cellular antioxidant capacity. Once formed, these species may engage in reactions to yield more potent oxidants that promote transition of the homeostatic vascular phenotype to a pathobiological state that is permissive for atherothrombogenesis. This dysfunctional vasculature is characterized by lipid peroxidation and aberrant lipid deposition, inflammation, immune cell activation, platelet activation, thrombus formation, and disturbed hemodynamic flow. Each of these pathobiological states is associated with an increase in the vascular burden of free radical species-derived oxidation products and, thereby, implicates increased oxidant stress in the pathogenesis of atherothrombotic vascular disease.     

Formation of native structure by intermolecular thiol-disulfide exchange reactions without oxidants in the folding of bovine pancreatic ribonuclease A

It has been shown previously that the oxidative folding of bovine pancreatic ribonuclease A proceeds through parallel pathways with two major native-like three-disulfide (3S) intermediates. We show here that, under some conditions, the native disulfide bonds can also be regenerated through disproportionation reactions; in other words, the protein can serve as its own redox reagent. The results also show that disulfide species of the unstructured 3S ensemble have a strong propensity to participate in intermolecular interactions. These interactions are favored at high protein concentration, temperature and pH, and lead to formation of the native structure during disulfide reshuffling in the rate-determining step.     

Energy Transduction Inside of Amphiphilic Vesicles: Encapsulation of Photochemically Active Semiconducting Particles

Amphiphilic bilayer membrane structures (vesicles) have been postulated to have been abiotically formed and spontaneously assemble on the prebiotic Earth, providing compartmentalization for the origin of life. These vesicles are similar to modern cellular membranes and can serve to contain water-soluble species, concentrate species, and have the potential to catalyze reactions. The origin of the use of photochemical energy in metabolism (i.e. energy transduction) is one of the central issues in the origin of life. This includes such questions as how energy transduction may have occurred before complex enzymatic systems, such as required by contemporary photosynthesis, had developed and how simple a photochemical system is possible. It has been postulated that vesicle structures developed the ability to capture and transduce light, providing energy for reactions. It has also been shown that pH gradients across the membrane surface can be photochemically created, but coupling these to drive chemical reactions has been difficult. Colloidal semiconducting mineral particles are known to photochemically drive redox chemistry. We propose that encapsulation of these particles has the potential to provide a source of energy transduction inside vesicles, and thereby drive protocellular chemistry, and represents a model system for early photosynthesis. In our experiments we show that TiO₂ particles, in the ~20 nm size range, can be incorporated into vesicles and retain their photoactivity through the dehydration/rehydration cycles that have been shown to concentrate species inside a vesicle.     

Detection of radicals produced by reaction of hydroperoxides with rat liver microsomal fractions.

EPR spin trapping using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 3,5-dibromo-4-nitrosobenzene sulphonic acid (DBNBS) has been employed to examine the generation of radicals produced on reaction of a number of primary, secondary and lipid hydroperoxides with rat liver microsomal fractions in both the presence and absence of reducing equivalents. Two major mechanisms of radical generation have been elucidated. In the absence of NADPH or NADH, oxidative degradation of the hydroperoxide occurs to give initially a peroxyl radical which in the majority of cases can be detected as a spin adduct to DMPO; these radicals can undergo further reactions which result in the generation of alkoxyl and carbon-centered radicals. In the presence of NADPH (and to a lesser extent NADH) alkoxyl radicals are generated directly via reductive cleavage of the hydroperoxide. These alkoxyl radicals undergo further fragmentation and rearrangement reactions to give carbon-centered species which can be identified by trapping with DBNBS. The type of transformation that occurs is highly dependent on the structure of the alkoxyl radical with species arising from beta-scission, 1,2-hydrogen shifts and ring closure reactions being identified; these processes are in accord with previous chemical studies and are characteristic of alkoxyl radicals present in free solution. Studies using specific enzyme inhibitors and metal-ion chelators suggest that most of the radical generation occurs via a catalytic process involving haem proteins and in particular cytochrome P-450. An unusual species (an acyl radical) is observed with lipid hydroperoxides; this is believed to arise via a cage reaction after beta-scission of an initial alkoxyl radical.     

Regio- and stereospecific analysis of glycerolipids

In recent years researchers have recognized the potential value of comprehensive lipid profiling (lipidomics), which was invented and promoted by lipidologists who recognized the many valuable applications that grew out of the fields of DNA profiling (genomics) and protein profiling (proteonomics). Through lipid class-selective intrasource ionization and subsequent analysis of two-dimensional cross-peak intensities, the chemical identity and mass composition of individual molecular species of most lipid classes can now be determined in a chloroform extract. There remains, however, the necessity to distinguish the enantiomers and isobaric regioisomers resulting from enzymatic and chemical reactions, which conventional high performance liquid chromatography/mass spectrometry (HPLC/MS) has been slow to accommodate, and tandem MS unable to provide. While reversed-phase HPLC can separate regioisomers, normal-phase HPLC can resolve diastereomers, and chiral-phase HPLC can effect dramatic resolution of enantiomers, the full potential of the combined systems has seldom been exploited. The present chapter calls attention to both recent and earlier combinations of these methodologies with mass spectrometry, which allows the HPLC/ESI (electrospray ionization)-MS/MS separation and identification of enantiomeric diacylglycerols, triacylglycerols, and glycerophospholipids as well as their isobaric regioisomers. These developments permit further expansion of lipid profiling (lipidomics) and better understanding of lipid metabolism.     

The effect of carotenoids on the concentration of singlet oxygen in lipid membranes

An effect of β-carotene and its polar derivative, zeaxanthin, on a concentration of singlet oxygen in lipid membranes was studied in a model system. The carotenoids were incorporated into the membranes of small unilamellar liposomes at a concentration of 0.15 mol% with respect to lipid. Singlet oxygen was generated in a liposome suspension via photosensitization of toluidine blue, and its concentration in a membrane was detected with application of a specific fluorescence probe (singlet oxygen sensor green reagent) located in the lipid bilayer. The results show the carotenoid-dependent decrease in the concentration of singlet oxygen in the membranes formed with unsaturated lipids (egg yolk phosphatidylcholine and digalactosyldiacylglycerol) but not in the case of the membranes formed with a saturated lipid (dimyristoylphosphatidylcholine). The effect of carotenoids was about twice as high as in the case of cholesterol present in liposomes at the same concentration. The results suggest that carotenoids protect membranes formed with unsaturated lipids against singlet oxygen through combined activity of different mechanisms: modification of structural properties of the lipid bilayers, physical quenching of singlet oxygen and chemical reactions leading to the pigment oxidation. The latter conclusion is based on the analysis of the absorption spectra of liposomes before and after light exposure. An importance of the different modes of protection by carotenoids against single oxygen toxicity towards biomembranes is discussed.     

Avoidance of oxidative-stress perturbation in yeast bioprocesses by proteomic and genomic biostrategies?

AIMS: Bioprocess oxidative stress caused by many reactive oxygen species (ROS) can lead to largely irreversible perturbation of yeast bioprocesses. These include the production of proteins derived from recombinant DNA yeast technology (aerobically grown Saccharomyces cerevisiae). These proteins include rennin, amyloglucosidases (glucamylases), interferons, interleukins, insulin, monoclonal antibodies, tissue plasminogen activators (t-PA), sexually transmitted disease antigens, and measles, mumps and rubella antigens, growth hormones, somatotropin, blood clotting factors VIII and XIII. In addition, there may be a demand for severe acute respiratory syndrome-coronavirus antigens, hepatitis A, B and C viral-selected antigens, HIV retroviral antigens, influenza antigens, trypanosomal antigens, and foot and mouth disease antigens. Prevention of oxidative stress has been achieved by application of antioxidant redox metalloenzymes such as superoxide dismutases (containing Cu/Zn cytosolic, Mn mitochondrial and Fe bacterial) glutathione peroxidases (and other Se-containing proteins and enzymes such as the thioredoxins), catalases (Fe-containing), cytochrome c peroxidases (Fe-containing), ceruloplasmins (Cu-containing), metallothionines (these cysteine thiol-rich proteins bind ions of cadmium and mercury) and tyrosinases(Cu-containing). METHODS AND RESULTS: ROS are generated inadvertently by single metal valency couples such as FeII/FeIII and by FeIII/FeV present in 2700 (including 57 human) isoforms in cytochromes P450 mixed-function oxidases (EC 1.14.14.1; O2 : mono-oxygenase NADPH/NADH requiring). In addition, mixed-metal couples such as valency unmatched forms in CuI/FeII and FeIII/MnIV can recycle electrons. Moreover, proteins/protein chaperone couples can recycle electrons, often where futile-recycling systems have been instigated. Furthermore, oxidized membrane phospholipids (R) can form ROOH (lipid hydroperoxides) and ROH (lipid alkoxides) that can generate ROS through Fenton chemistry (iron-catalysed) chain reactions. Utilization of chain-breaking antioxidants such as vitamin E (alpha-tocopherol) in the lipid phase and vitamin C (ascorbate) in the aqueous phase can terminate these ROS-producing reactions. CONCLUSIONS: The main significance of the study is that proteomic strategies of relief from bioprocess perturbation by ROS of yeast fermentations (used to manufacture proteins required in the food and therapeutic bioindustries) may become possible through addition of selected proteins (including metalloenzymes). The main impact of the study is that the utilization of genetically modified (GM) yeast produced by recombinant DNA technology genomic strategies could circumvent the bioprocessing problems that otherwise result from the bioprocess perturbations: this is as a result of oxidative stress caused by ROS, which is avoidable by deployment of appropriate antioxidants such as vitamins E, C and D (and antioxidant proteins and enzymes often of microbial origin via recombinant DNA technology).     

Free radical biology and medicine: it's a gas, man!

We review gases that can affect oxidative stress and that themselves may be radicals. We discuss O(2) toxicity, invoking superoxide, hydrogen peroxide, and the hydroxyl radical. We also discuss superoxide dismutase (SOD) and both ground-state, triplet oxygen ((3)O(2)), and the more energetic, reactive singlet oxygen ((1)O(2)). Nitric oxide ((*)NO) is a free radical with cell signaling functions. Besides its role as a vasorelaxant, (*)NO and related species have other functions. Other endogenously produced gases include carbon monoxide (CO), carbon dioxide (CO(2)), and hydrogen sulfide (H(2)S). Like (*)NO, these species impact free radical biochemistry. The coordinated regulation of these species suggests that they all are used in cell signaling. Nitric oxide, nitrogen dioxide, and the carbonate radical (CO(3)(*-)) react selectively at moderate rates with nonradicals, but react fast with a second radical. These reactions establish "cross talk" between reactive oxygen (ROS) and reactive nitrogen species (RNS). Some of these species can react to produce nitrated proteins and nitrolipids. It has been suggested that ozone is formed in vivo. However, the biomarkers that were used to probe for ozone reactions may be formed by non-ozone-dependent reactions. We discuss this fascinating problem in the section on ozone. Very low levels of ROS or RNS may be mitogenic, but very high levels cause an oxidative stress that can result in growth arrest (transient or permanent), apoptosis, or necrosis. Between these extremes, many of the gasses discussed in this review will induce transient adaptive responses in gene expression that enable cells and tissues to survive. Such adaptive mechanisms are thought to be of evolutionary importance.     

Non-enzymatically derived minor lipids found in Escherichia coli lipid extracts

Electrospray ionization mass spectrometry is a powerful technique to analyze lipid extracts especially for the identification of new lipid metabolites. A hurdle to lipid identification is the presence of solvent contaminants that hinder the identification of low abundance species or covalently modify abundant lipid species. We have identified several non-enzymatically derived minor lipid species in lipid extracts of Escherichia coli; phosphatidylmethanol, ethyl and methyl carbamates of PE and N-succinyl PE were identified in lipid extracts of E. coli. Phosphatidylmethanol (PM) was identified by exact mass measurement and collision induced dissociation tandem mass spectrometry (MS/MS). Extraction in the presence of deuterated methanol leads to a 3 atomic mass unit shift in the [M-H](-) ions of PM indicating its formation during extraction. Ethyl and methyl carbamates of PE, also identified by exact mass measurement and MS/MS, are likely to be formed by phosgene, a breakdown product of chloroform. Addition of phosgene to extractions containing synthetic PE significantly increases the levels of PE-MC detected in the lipid extracts by ESI-MS. Extraction in the presence of methylene chloride significantly reduced the levels of these lipid species. N-succinyl PE is formed from reaction of succinyl-CoA with PE during extraction. Interestingly N-succinyl PE can be formed in an aqueous reaction mixture in the absence of added E. coli proteins. This work highlights the reactivity of the amine of PE and emphasizes that careful extraction controls are required to ensure that new minor lipid species identified using mass spectrometry are indeed endogenous lipid metabolites.