Kinetic products of linoleate peroxidation: rapid beta-fragmentation of nonconjugated peroxyls.
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Antioxidants↗
Biomedical subjects
N A Porter
Publications and source records attributed to N A Porter.
Minimizing tocopherol-mediated radical phase transfer in low-density lipoprotein oxidation with an amphiphilic unsymmetrical azo initiator.
The antioxidant alpha-tocopherol (alpha-TOH) has been found to act as a pro-oxidant under many in vitro conditions. The observed tocopoherol-mediated peroxidation (TMP) is dependent on two primary factors. (1) Chain transfer: alpha-TO. radical reacts with lipid to form lipid peroxyl radicals. (2) Phase transfer: alpha-TOH can transport radical character into the lipoprotein. Given the limitations of existing initiators, there is a need for new compounds that avoid the requirement for alpha-TOH to act as a phase-transfer agent. We report here a study showing that the new unsymmetrical azo compound, C-8, initiates LDL lipid peroxidation without requirement for alpha-TOH. This initiator provides a steady source of free amphiphilic peroxyl radicals that efficiently initiates oxidation of alpha-TOH-depleted LDL at a rate comparable to that reported for the very reactive hydroxyl radical (.OH). With other initiators tested, unsymmetrical C-12 and C-16 and symmetrical C-0 and MeOAMVN, alpha-TOH-depleted LDL displayed significant resistance to oxidation. Results indicate that the amphiphilic nature of the unsymmetrical initiators increases their partitioning into lipoprotein depending on the hydrocarbon chain length, and the symmetrical azo initiators C-0 and MeOAMVN primarily remain in the aqueous phase. Evidence suggests that even when the phase-transfer activity of alpha-TOH is limited, with the use of an initiator such as C-8, the mechanism of peroxidation remains controlled by TMP chain-transfer activity.
Azo Compounds↗
Two distinct pathways of formation of 4-hydroxynonenal. Mechanisms of nonenzymatic transformation of the 9- and 13-hydroperoxides of linoleic acid to 4-hydroxyalkenals.
The mechanism of formation of 4-hydroxy-2E-nonenal (4-HNE) has been a matter of debate since it was discovered as a major cytotoxic product of lipid peroxidation in 1980. Recent evidence points to 4-hydroperoxy-2E-nonenal (4-HPNE) as the immediate precursor of 4-HNE (Lee, S. H., and Blair, I. A. (2000) Chem. Res. Toxicol. 13, 698-702; Noordermeer, M. A., Feussner, I., Kolbe, A., Veldink, G. A., and Vliegenthart, J. F. G. (2000) Biochem. Biophys. Res. Commun. 277, 112-116), and a pathway via 9-hydroperoxylinoleic acid and 3Z-nonenal is recognized in plant extracts. Using the 9- and 13-hydroperoxides of linoleic acid as starting material, we find that two distinct mechanisms lead to the formation of 4-H(P)NE and the corresponding 4-hydro(pero)xyalkenal that retains the original carboxyl group (9-hydroperoxy-12-oxo-10E-dodecenoic acid). Chiral analysis revealed that 4-HPNE formed from 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13S-HPODE) retains >90% S configuration, whereas it is nearly racemic from 9S-hydroperoxy-10E,12Z-octadecadienoic acid (9S-HPODE). 9-Hydroperoxy-12-oxo-10E-dodecenoic acid is >90% S when derived from 9S-HPODE and almost racemic from 13S-HPODE. Through analysis of intermediates and products, we provide evidence that (i) allylic hydrogen abstraction at C-8 of 13S-HPODE leads to a 10,13-dihydroperoxide that undergoes cleavage between C-9 and C-10 to give 4S-HPNE, whereas direct Hock cleavage of the 13S-HPODE gives 12-oxo-9Z-dodecenoic acid, which oxygenates to racemic 9-hydroperoxy-12-oxo-10E-dodecenoic acid; by contrast, (ii) 9S-HPODE cleaves directly to 3Z-nonenal as a precursor of racemic 4-HPNE, whereas allylic hydrogen abstraction at C-14 and oxygenation to a 9,12-dihydroperoxide leads to chiral 9S-hydroperoxy-12-oxo-10E-dodecenoic acid. Our results distinguish two major pathways to the formation of 4-HNE that should apply also to other fatty acid hydroperoxides. Slight ( approximately 10%) differences in the observed chiralities from those predicted in the above mechanisms suggest the existence of additional routes to the 4-hydroxyalkenals.
Aldehyde-Lyases↗
Separation and identification of phospholipid peroxidation products.
The molecular species in mixtures of phospholipid hydroperoxides are difficult to separate and identify by typical chromatographic and mass spectrometric techniques. As reported by Havrilla and coworkers, silver ion coordination ion-spray mass spectrometry (CIS-MS) has proven to be a powerful technique for the identification of mixtures of hydroperoxides. This ionization technique, which involves the formation of Ag+ adducts of the hydroperoxides, provides valuable, unambiguous structural information about the hydroperoxides. Herein, we report a method for the analysis and identification of phospholipid hydroperoxides using CIS-MS. We also report an improved method for the separation of phospholipid hydroperoxides by reversed-phase high-performance liquid chromatography (RP-HPLC), which, for the first time, separates some of the hydroperoxide isomers. CIS-MS can be coupled with this RP-HPLC method by the addition of AgBF4 to the mobile phase or to the HPLC effluent postcolumn, thus allowing powerful HPLC-MS techniques to be used to identify complex mixtures of phospholipid hydroperoxides.
Arachidonic Acid↗
Analysis of diacyl peroxides by Ag+ coordination ionspray tandem mass spectrometry: free radical pathways of complex decomposition.
Organic peroxides have significance in organic synthesis and biological processes. Characterization of these compounds with weak O-O bonds is sometimes difficult due to their thermal instability and sensitivity to acid or base. Coordination of diacyl peroxides with AgBF4 provides a means for analysis of these compounds by coordination ionspray tandem mass spectrometry (CIS-MS/MS). Precursor ion (Q1) scans of acetyl benzoyl peroxide give two Ag+ adducts, [M + Ag + solvent]+ and [M + Ag + M]+. These silver ion adducts can be selectively dissociated (CID) to give unique structural information about the analyte. Decomposition of the [M + Ag + solvent]+ adduct generates fragmentation products due to apparent homolytic cleavage of the O-O bond followed by decarboxylation of the resultant radicals. The bis-diacylperoxide complex, [M + Ag + M]+ gives CID pathways that involve homolysis of the (O-O bond and free radical cross-coupling of the two diacyl peroxides coordinated to the silver ion, i.e. formation of dibenzoyl peroxide, phenyl benzoate, and biphenyl from acetyl benzoyl peroxide. The observation of free radical CID modes is uncommon in mass spectrometry but these pathways are consistent with well-known solution and gas phase processes for peroxide compounds. The proposed fragmentation pathways have been supported by experiments with (18)O and deuterated substrates. This technique can be applied to analyze diacyl peroxides with different substituents as well.
Chromatography, Thin Layer↗
Structural analysis of diacyl peroxides by electrospray tandem mass spectrometry with ammonium acetate: bond homolysis of peroxide-ammonium and peroxide-proton adducts.
Organic peroxides have significant implications in organic chemistry and biological processes. The weak O-O bond makes them extremely difficult to characterize by conventional analytical methods. Diacyl peroxides are one of the major radical sources in polymerization and organic synthesis. It is well known that diacyl peroxides are thermal labile and thus are not amenable to study by gas chromatography/mass spectrometry (GC/MS). Electrospray tandem mass spectrometry (ESI-MS/MS) has been applied to the structural analysis of diacyl peroxides by formation of ammonium adducts. Collision induced dissociation (CID) studies of the ammonium adducts of the peroxide [M + NH(4)](+) give collision energy dependent fragments. For most diacyl peroxides, homolysis of the peroxy bond predominates the fragmentation pathways of the peroxide-ammonium adducts. Deuterated substrates have been employed to provide evidence for typical fragmentation pathways. The CID studies were also used to locate the O-18 in some O-18 specifically labeled diacyl peroxides. For branched alkyl or alkoxy substrates, McLafferty rearrangement and decarboxylation become a major pathway. By comparison with some anhydride analogues, ESI-MS/MS can also be used to study this class of compounds.
Mass Spectrometry↗
Synthesis, structure, and thermal properties of 1,2-dipalmitoylgalloylglycerol (DPGG), a novel self-adhering lipid.
A novel diacyl glycerol-based lipid with a polyphenolic head group has been synthesized and characterized. X-ray diffraction experiments show that this lipid, 1,2-dipalmitoylgalloylglycerol (DPGG), hydrates to form gel phase bilayers at 20 degrees C with extremely narrow interbilayer fluid separations, indicating that apposing DPGG bilayers strongly adhere to each other. Differential scanning calorimetry shows that fully hydrated DPGG exhibits a pretransition exotherm (3.7 kcal/mol) at 52 degrees C and a high enthalpy (11.3 kcal/mol) main endothermic transition at 69 degrees C. These thermal properties are similar to those of galactosylceramides with similar hydrocarbon chain compositions. The adhesive and thermal properties of DPGG are likely due to both intermolecular hydrogen-bonding and hydrophobic interactions between the aromatic rings on the gallic acids.
Calorimetry, Differential Scanning↗
Design of unsymmetrical azo initiators to increase radical generation efficiency in low-density lipoproteins.
Lipid peroxidation studies often employ the use of azo initiators to produce a slow, steady source of free radicals, but the lack of initiators capable of efficiently generating radicals in lipid regions has created persistent problems in these investigations. For example, experiments with symmetrical lipophilic or symmetical hydrophilic azo initiators increasingly suggest that their initiation mechanisms in low-density lipoproteins (LDL) rely upon the presence of alpha-tocopherol to mediate peroxidation. We report here the synthesis and study of the new unsymmetrical azo compounds SA-1, SA-2, C-16, C-12, and C-8 that decompose over a range of convenient temperatures and improve radical generation efficiency and access to lipid compartments. The half-life for decomposition (tau(1/2)) of the unsymmetrical initiators at 37 degrees C in methanol covered a range of 121 hours for SA-1, 77 hours for SA-2, and approximately 25 hours for the series C-16, C-12, and C-8. Agarose gel electrophoresis of LDL incubated with these unsymmetrical initiators supports the conclusion that the initiators associate with lipoprotein without disrupting integrity of the particle. The unsymmetical initiator C-8 when compared to symmetical hydrophilic initiator C-0 is capable of providing increased peroxidation of LDL, as monitored by formation of cholesteryl linoleate oxidation products and consumption of alpha-tocopherol. Efficiency of radical generation in lipophilic and hydrophilic compartments was found to be represented with the use of the radical scavenger combination alpha-tocopherol and uric acid, but not with the use of N,N'-Diphenyl-p-phenylenediamine (DPPD) and uric acid. These unsymmetrical initiators, when compared to the widely used symmetrical azo initiators, provide an advantage of free radical production, lipophilic access, and constant radical generation in the investigation of lipid peroxidation in low-density lipoproteins.
Animals↗
Polyphenols increase adhesion between lipid bilayers by forming interbilayer bridges.
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Flavonoids↗
Photo-reversible binding of thrombin to avidin by means of a photolabile inhibitor.
Human alpha-thrombin and Factor Xa were acylated at their active site serine hydroxyls with biotin-substituted cinnamate derivatives 1b-1c. These acyl enzymes (2) showed no enzyme activity in the absence of light. On irradiation with light of wavelength 366 nm for 6 min, however, up to 80% of pre-inhibition activity was regained. This photo-deacylation of the modified enzyme results in the formation of active enzyme and a coumarin by-product (3). In addition, the acyl enzyme that results from incubation of 1c with thrombin was capable of binding to avidin, both immobilized and free in solution. Furthermore, the complex formed between the thrombin acyl enzyme (2c) and avidin was capable of binding to immobilized biotin.
Acylation↗
In vivo photoactivation of caged-thrombin.
Aberrant ocular neovascularization is a major cause of blindness in the world. Abnormal blood vessels in the eye may produce corneal opacification, corneal transplant rejection, neovascular glaucoma, vitreous hemorrhage, traction retinal detachment, and subretinal scars from choroidal neovascular membranes (1-5). Light-induced clotting of blood within these abnormal vessels could provide a novel method for the ablation of deleterious neovascularization. Thrombin is a serine proteinase that participates in the final stages of the coagulation cascade. An inhibitor of thrombin, p-Amidinophenyl-(E)-4-diethylamino-2-hydroxy-alpha-methylcinnamate hydrochloride, MeCINN (1), covalently attaches to the active site serine hydroxyl, inhibiting or caging, the enzyme. Photolysis of the caged-thrombin in vitro causes a trans-cis isomerization of MeCINN which leads to regeneration of active enzyme and cleaving of fibrinogen into fibrin (6). Using a rabbit model of corneal neovascularization, we found that light at 366 nm safely and effectively photoactivates intravenous caged-thrombin and produces localized thrombosis in vivo. These results suggest that intravascular photoactivation of caged-thrombin could be used to occlude abnormal blood vessels in the human eye.
Animals↗
The interaction of polyphenols with bilayers: conditions for increasing bilayer adhesion.
Because proteins and other molecules with a high polyphenol content are commonly involved in adhesion processes, we are investigating the interactions between polyphenols and biological materials. A naturally occurring polyphenol that binds a variety of proteins and lipids is tannic acid (TA), which contains five digallic acid residues covalently linked to a central D-glucose. A previous study has shown that TA increases the adhesion between apposing phosphatidylcholine (PC) bilayers and over a very narrow concentration range collapses the interbilayer fluid space from about 15 A to 5 A. To determine the chemical requirements a polyphenolic molecule must possess to increase bilayer adhesion, we have synthesized several simpler TA analogs that vary in their size, shape, and number of gallic acid and hydroxyl groups. X-ray diffraction, absorbance, binding, and differential scanning calorimetry measurements were used to investigate the interaction of these polyphenolic molecules with egg PC (EPC) and dipalmitoyl PC (DPPC) bilayers. Of these synthetic polyphenols, only penta-O-galloyl-alpha-D-glucose (PGG) was able to completely mimic the effects of TA by collapsing the interbilayer fluid space from 15 A to 5 A, decreasing the dipole potential by about 300 mV, increasing the transition enthalpy of DPPC liposomes, and inducing an interdigitated phase in DPPC. Binding studies indicated that the fluid space was reduced to 5 A at an EPC:PGG mole ratio of 5:1. We conclude that these polyphenols collapse the fluid space of PC bilayers because they 1) are amphipathic and partition into the bilayers interfacial region, 2) are long enough to span the interbilayer space, 3) contain several gallic acids distributed so that they can partition simultaneously into apposing bilayers, and 4) have sufficient gallic acid residues to interact with all lipid headgroups and cover the bilayer surface. Under these conditions we conclude that the polyphenols from interbilayer bridges. We argue that these bridges are stabilized by increased adhesion arising from an increased van der Waals interaction between apposing bilayers, electrostatic interactions between the pi electrons in the phenol ring and the -(N+CH3)3 groups on the PC headgroups, decreased hydration repulsion between bilayers, and hydrogen bonds between the H-bond-donating moieties on the polyphenols and H-bond-accepting groups in the bilayer.
1,2-Dipalmitoylphosphatidylcholine↗
Identification and quantification of regioisomeric cholesteryl linoleate hydroperoxides in oxidized human low density lipoprotein and high density lipoprotein.
Oxidation of human LDL is implicated as an initiator of atherosclerosis. Isolated low density lipoprotein (LDL) and high density lipoprotein (HDL2) were exposed to aqueous radicals generated from the thermolabile azo compound 2,2'-azobis(2-amidinopropane) dihydrochloride. The primary nonpolar lipid products formed from the autoxidation of LDL and HDL were the regioisomeric cholesteryl linoleate hydroperoxides. In LDL oxidations, 9- and 13-hydroperoxides with trans,cis conjugated diene were formed as the major oxidation products if endogenous alpha-tocopheral was present in the LDL. After extended oxidation of LDL, at the time when endogenous alpha-tocopherol was consumed, the two trans,cis conjugated diene hydroperoxides began to disappear and the 9- and 13-hydroperoxides with trans,trans conjugated diene appeared. At very long oxidation times, none of the primary products, the conjugated diene hydroperoxides, were present. In HDL2, which has only very low levels of antioxidants, both the 9- and 13-hydroperoxides with trans,cis conjugated diene and the 9- and 13-hydroperoxides with trans,trans conjugated diene were formed at early stages of oxidation. The corresponding alcohols were also formed in the HDL2 oxidations. A mechanistic hypothesis consistent with these observations is presented.
Alcohols↗
Mechanisms of free radical oxidation of unsaturated lipids.
The primary products formed from the autoxidation of lipids can be understood based upon a mechanism that involves five different reaction types. These reactions are: reaction of a carbon radical and molecular oxygen, atom transfer of a hydrogen from substrate to the chain carrying peroxyl, fragmentation of the chain carrying peroxyl to give oxygen and a carbon radical, rearrangement of the peroxyl, and cyclization of the peroxyl. The mechanism of these primary reaction steps has been the focus of extensive research over the past fifty years, and the current level of understanding of these transformations is the subject of this review.
Free Radicals↗
Suggested mechanisms for the production of 4-hydroxy-2-nonenal from the autoxidation of polyunsaturated fatty acids.
Autoxidation of certain polyunsaturated fatty acids (PUFA) produces 4-hydroxy-2-nonenal (HNE). Of all of the many products produced during PUFA autoxidation, HNE is one of the more interesting. It is remarkably cytotoxic, and appears to play a role in certain types of pathology. This manuscript suggests, for the first time, mechanisms that rationalize the production of HNE from PUFA that undergo autoxidation. The mechanisms require, in agreement with the facts, an n-6 fatty acid and iron catalysis.
Aldehydes↗
Photocoagulation of human plasma: acyl serine proteinase photochemistry.
Human alpha-thrombin or bovine Factor Xa was acylated at the active site serine hydroxyl with alpha-methyl-2-hydroxy-4-diethylaminocinnamic acid. These modified serine proteinase enzymes showed no plasma coagulation biological activity in the absence of light. Photolysis of the acyl serine proteinase enzymes in plasma for 1-35 s with monochromatic 366 nm light isolated from a high pressure mercury arc results in coagulation of the plasma. For example, photolysis of 3 NIH U of the acyl human alpha-thrombin for 5 s in human plasma results in a clot in 23 s. For comparison, 1 NIH U of unmodified human alpha-thrombin gave a clot in 21 s under the conditions of the assay but without photolysis. Appropriate controls showed that the coagulation is the result of the formation of active thrombin due to photodeacylation of the enzymes. The photoinduced clotting time measured is dependent on acyl thrombin concentration and photolysis time. Thus higher concentrations of acyl thrombin and longer photolysis times give a shorter clotting time. A kinetic scheme based upon Lineweaver-Burke analysis of the clotting process is developed.
Acylation↗
Evaluation of p-amidinophenyl esters as potential antithrombotic agents.
Three p-amidinophenyl esters have been synthesized and characterized as irreversible inhibitors of the vitamin-K dependent proteinases; factors IXa, Xa and thrombin (Turner et al. [4]).+ In the present report we describe the in vitro and in vivo effects of these agents on standard coagulation tests in vitro and in blood from animals treated with the compounds. At a concentration of 500 microM, the three esters increased the activated partial thromboplastin time (PTT) of pooled human plasma 3 to 5-fold. The prothrombin time increased 1.4 to 3.7-fold under similar conditions. The p-amidinophenyl ester of cinnamic acid (CINN) showed the most pronounced effect on both assays. This ester also is the best inhibitor of human factors IXa and Xa, while the p-amidinophenyl ester of benzoic acid (BENZ) is a slightly better alpha-thrombin inhibitor (4). The effect of these esters on the thrombin clotting time correlated with in vitro kinetic measurements of alpha-thrombin inhibition rates. Both BENZ and CINN increased the assay endpoint more than 6-fold. The three esters also were studied using mouse plasma. A comparable effect on the PTT was noted. Intravenous administration of 300 microliter of 1 mM CINN as a single bolus in mice caused a 2.3-fold increase in the PTT which remained 1.2-fold normal 2 h later. The BENZ and alpha-methyl-cinnamic acid (MECINN) esters were somewhat less effective as predicted from their in vitro effect on the PTT. This investigation and previous studies indicate that these compounds demonstrate low toxicity at therapeutic levels. It is concluded that the p-amidinophenyl esters may be useful in antithrombotic therapy.
Amidines↗
p-Amidino esters as irreversible inhibitors of factors IXa and Xa and thrombin.
A number of inhibitors of thrombin and factor Xa have been described; however, only one inhibitor of factor IXa has been reported. This compound, dansyl-Glu-Gly-Arg chloromethyl ketone (DEGER), inhibits porcine factor IXa with a second-order rate constant of 2.2 X 10(4) M-1 min-1. We now describe the synthesis and characterization of three p-amidinophenyl esters that inhibit human factor IXa with second-order rate constants comparable to those observed with human and bovine factor Xa and alpha-thrombin. These rate constants of inhibition, moreover, are 2-30-fold greater than observed when DEGER is employed to inhibit porcine factor IXa. Additional advantages of these derivatives include their ease of synthesis and low degree of toxicity. The p-amidinophenyl ester of benzoic acid was employed to inhibit human factor IXa, and the plasma clearance of the protein was studied in mice. These experiments demonstrate for the first time that the endothelial binding previously reported with factor IXa is independent of the active site, a finding similar to the behavior observed with factor Xa and alpha-thrombin in this and previous reports.
Acylation↗