Acid Oxidation Products Research Articles

Formation of Hydroxyl Radicals in the Human Lens is Related to the Severity of Nuclear Cataract

Recent studies have identified specific hydroxylated amino acid oxidation products which strongly suggest the presence of hydroxyl radical (HO·)-damaged proteins in human cataractous lenses. In the present study, the ability of early stage (type II) and advanced (type IV) nuclear cataractous lens homogenates to catalyse HO· production in the presence of H2O2was investigated using electron paramagnetic resonance (EPR) spectroscopy with the free radical trap, 5,5-dimethyl-1-pyrroline- N -oxide (DMPO). Cataractous lens homogenates incubated with 1 m m H2O2generated a distinct HO· signal, which was significantly more intense in the nuclear region of the type IV compared to the type II lenses. The ability of individual lens nuclei and cortices to stimulate HO· production was positively correlated. The DMPO-HO· signal was competitively inhibited by ethanol, confirming that the DMPO-HO· signal was due to HO· formation and not DMPO-OOH degradation. The metal ion chelator, diethylenetriaminepentaacetic acid, also inhibited HO· formation, indicating that lenticular metal ions play a key role in HO· formation. Cataractous lens homogenates also stimulated ascorbyl radical production, further suggesting the presence of redox-active metal ions in the tissue. Analysis of lenses for total Fe and Cu (using atomic absorption spectrometry) showed that the more advanced type IV lenses tended to have higher Fe, but similar Cu, levels compared to the type II lenses. The levels of both metals were lower in non-cataractous lenses. These data support the hypothesis that transition metal-mediated HO· production may play a role in the aetiology of age-related nuclear cataract.

Simultaneous determination of five oxidative DNA lesions in human urine.

A method, involving a HPLC prepurification followed by a GC/MS analysis, has been set up for the measurement of nucleic acid oxidation products in human urine. For this purpose, isotopically labeled internal standards have been prepared and used for isotope dilution mass spectrometric detection. Using this approach, four oxidized DNA bases, i.e., 5-hydroxyuracil, 5-(hydroxymethyl)uracil, 8-oxo-7,8-dihydroadenine, and 8-oxo-7,8-dihydroguanine, together with 8-oxo-7,8-dihydro-2'-deoxyguanosine have been simultaneously quantified in human urine samples. The levels of the oxidized nucleic acid constituents, as expressed in picomoles per milliliter, were determined to be, in decreasing order: 8-oxo-7,8-dihydroguanine (583 +/- 376) > 5-(hydroxymethyl)uracil (121 +/- 56) > 5-hydroxyuracil (58 +/- 23) > 8-oxo-7,8-dihydro-2'-deoxyguanosine (30 +/- 15) > 8-oxo-7,8-dihydroadenine (7 +/- 4). Attempts to determine the amount of 5,6-dihydroxy-5,6-dihydrothymine, 5-hydroxycytosine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine using the above HPLC-GC/MS method were unsuccessful.

Mass spectrometric quantification of amino acid oxidation products in proteins: insights into pathways that promote LDL oxidation in the human artery wall.

Oxidatively damaged low density lipoprotein (LDL) may play an important role in atherogenesis, but the physiologically relevant pathways have proved difficult to identify. Mass spectrometric quantification of stable compounds that result from specific oxidation reactions represents a powerful approach for investigating such mechanisms. Analysis of protein oxidation products isolated from atherosclerotic lesions implicates tyrosyl radical, reactive nitrogen species, and hypochlorous acid in LDL oxidation in the human artery wall. These observations provide chemical evidence for the reaction pathways that promote LDL oxidation and lesion formation in vivo.--Heinecke, J. W. Mass spectrometric quantification of amino acid oxidation products in proteins: insights into pathways that promote LDL oxidation in the human artery wall.

Concise review of the glutathione S-transferases and their significance to toxicology.

The glutathione S-transferases (GSTs; E.C. 2.5.1.18) are a multi-gene family of enzymes involved in the detoxification, and, in a few instances, activation of a wide variety of chemicals (Table 1). Since their discovery in the early 1970s, thousands of articles have been written on the structure, function, and toxicologic significance of GSTs, and it is obviously beyond the scope of this article to provide a comprehensive review of the field. This article will highlight the major features of this multi-gene family of enzymes, with a focus on human soluble (cytosolic) GSTs. A relatively comprehensive review of the GSTs was published in 1995, and provides an excellent source for detailed information and references about nearly all aspects of GSTs up to that time (Hayes and Pulford, 1995). (Because of space limitations, the present mini-review is not fully referenced, but it does include some newer references not covered in previous reviews.) GSTs catalyze the nucleophilic attack of glutathione (GSH) on electrophilic substrates, thereby decreasing their reactivity with cellular macromolecules (Armstrong, 1997). These proteins have been found in virtually every living species examined, including plants, animals, and bacteria (Hayes and Pulford, 1995). Most GSTs exist as soluble enzymes, although a small family of microsomal GSTs has been characterized (Andersson et al., 1994; Jakobsson et al., 1996), and a mitochondrial GST, referred to as GST Kappa, has also been identified (Pemble et al., 1996). In humans and in rodents, the soluble GSTs are collectively expressed in rather large amounts, constituting as much as 4% of total soluble protein in the liver. Although no definitive physiological role for GSTs has yet been identified, several reactive endogenous molecules, including ab-unsaturated keto prostaglandins (e.g., PGA2, PGJ2), and endogenous fatty acid oxidation products, such as 4-hydroxy-2-nonenal, serve as substrates for certain GSTs (Hayes and Pulford, 1995) (Table 2). One human GST (hGSTM2-2) has recently been found to exhibit unique and high catalytic activity toward reactive quinones of endogenous catecholamines (e.g., dopaminochrome, formed by oxidation of dopamine), and thus may play some protective role against endogenous oxidative tissue damage in the brain (Baez et al., 1997). The soluble forms exist as dimeric proteins, with subunit molecular weights of approximately 25 kDa (Table 1). Each subunit of the dimeric enzyme has an active site composed of 2 distinct functional regions: a hydrophilic G-site, which binds the physiological substrate glutathione, and an adjacent H-site which provides a hydrophobic environment for the binding of structurally diverse electrophilic substrates (Armstrong, 1997). The G-site is highly conserved between all GSTs due to its high specificity for GSH, whereas the H-site can be quite divergent between different GSTs, and exhibits broad and variable substrate binding specificity.

13-(S)-Hydroxyoctadecadienoic acid (13-HODE) incorporation and conversion to novel products by endothelial cells

13(S)-Hydroxy-[12,13-3H]octadecadienoic acid (13-HODE), a linoleic acid oxidation product that has vasoactive properties, was rapidly taken up by bovine aortic endothelial cells. Most of the 13-HODE was incorporated into phosphatidylcholine, and 80% was present in the sn-2 position. The amount of 13-HODE retained in the cells gradually decreased, and radiolabeled metabolites with shorter reverse-phase high-performance liquid chromatography retention times (RT) than 13-HODE accumulated in the extracellular fluid. The three major metabolites were identified by gas chromatography combined with mass spectrometry as 11-hydroxyhexadecadienoic acid (11-OH-16:2), 9-hydroxytetradecadienoic acid (9-OH-14:2), and 7-hydroxydodecadienoic acid (7-OH-12:2). Most of the radioactivity contained in the cell lipids remained as 13-HODE. However, some 11-OH-16:2 and several unidentified products with longer RT than 13-HODE were detected in the cell lipids. Normal human skin fibroblasts also converted 13-HODE to the three major chain-shortened metabolites, but Zellweger syndrome fibroblasts produced only a very small amount of 11-OH-16:2. Therefore, the chain-shortened products probably are formed primarily by peroxisomal β-oxidation. These findings suggest that peroxisomal β-oxidation may constitute a mechanism for the inactivation and removal of 13-HODE from the vascular wall. Because this is a gradual process, some 13-HODE that is initially incorporated remains in endothelial phospholipids, especially phosphatidylcholine. This may be the cause of some of the functional perturbations produced by 13-HODE in the vascular wall.—Fang, X., T. L. Kaduce, and A. A. Spector. 13-(S)-Hydroxyoctadecadienoic acid (13-HODE) incorporation and conversion to novel products by endothelial cells. J. Lipid Res. 1999. 40: 699–707.

Role of lipid peroxidation products in the formation of advanced glycation end products: Anin vitro study on collagen

This report provides a new possible link between increased lipid peroxidation and tissue damage in diabetes. Advanced Glycation End products (AGEs) have been well established as one of the major factors responsible for multiorgan damage seen in diabetes and aging. We report here that the interaction between lipid peroxidation products and the long-lived structural protein, collagen, results in the formation of products that are structurally or immunologically similar to AGEs. Enzyme-linked immunoassays revealed crossreactivity between anti-AGE antibodies and collagen incubated with linoleic acid/arachidonic acid for 4 weeks under oxidative conditions. Formation of AGEs from lipid oxidation products and collagen was further confirmed by the inhibitory effect of aminoguanidine. Similarly, incubation of collagen with an end product of lipid peroxidation, malondialdehyde (MDA), resulted in the formation of AGEs. It was also noted that the incubation of collagen with equimolar concentrations of glucose or linoleic acid/arachidonic acid resulted in the formation of almost similar amount of AGEs. Another interesting observation was that the level of AGEs correlated well with the crosslinking of collagen (r=0.93) and also with the intensity of fluorescence (ex: 390 nm/em: 460 nm) (r=0.99). However the rate of fluorescence development was observed at a faster rate than the formation of AGEs when the concentration of MDA was increased from 5 mM to 25/50 mM. Free radical scavengers failed to prevent lipid peroxidation-induced formation of AGEs. These results suggest that, in addition to reducing sugars, fatty acid oxidation products can also lead to the formation of products that are structurally similar to AGEs. The work presents a possible link between lipid peroxidation and diabetic complications, through AGEs. This study also suggests that the formation of AGEs is a possible explanation for lipid peroxide-induced crosslinking of collagen.

Determination of polar terpene oxidation products in aerosols by liquid chromatography–ion trap mass spectrometry

A new method for the determination of polar terpene oxidation products in secondary aerosols is described. It is based on collection of particles on PTFE filters and extraction with dichloromethane followed by analysis with liquid chromatography–ion-trap mass spectrometry (MS n ) using pneumatically assisted electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) with the ion-trap operated in the product scan mode. Separations were achieved on a C 18 reversed-phase column with methanol–water (0.1% acetic acid) as eluent. The method has a high sensitivity (instrument detection limit 0.7–7 pg/μl at S/N=3) and precision (5–10%) and a good linearity. Acidic oxidation products produce strong signals with ESI. They appear as negative quasimolecular ions [M−H] −, acetate adducts [M+CH 3COO] − and molecular clusters [2M−H] −. In the MS 2 mode these acids show strong signals from neutral loss of CO 2: [M−H−CO 2] −, and/or weaker signals from loss of H 2O: [M−H−H 2O] −, [M−H−H 2O−CO 2] −. Mass spectra were recorded by APCI for a number of oxygenated terpenoid standards containing keto groups, hydroxy groups, aldehyde groups, or epoxy groups. These compounds give intense signals as their positive quasimolecular ions [M+H] +, methanol adducts [M+H+CH 3OH] + and fragments from loss of water, such as [M+H−H 2O] +, [M+H+CH 3OH−H 2O] + and [M+H−2H 2O] +. By MS 2 and MS 3 neutral loss of H 2O and 2H 2O is observed. The method has been tested in analysis of aerosol from O 3–α-pinene and O 3–myrtenol and has been proved to compare well with classical methods based on derivatisation and gas chromatography mass spectrometry. Three new compounds, tentatively identified in aerosol are reported here for the first time: 10-hydroxypinonic acid, 9-hydroxynorpinonic acid and pinalic 4-acid.

Separation of linoleic acid oxidation products by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis

Separation of linoleic acid oxidation products by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis

Separation of linoleic acid oxidation products by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis.

In this work the suitability of micellar electrokinetic capillary chromatography (MEKC) and nonaqueous capillary electrophoresis (CE) to the analysis of the primary oxidation products of linoleic acid was studied with uncoated fused-silica capillaries. The primary autoxidation products of linoleic acid are the four hydroperoxide isomers 13-hydroperoxy-cis-9, trans-11-octadecadienoic acid, 13-hydroperoxy-trans-9, trans-11-octadecadienoic acid, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid, 9-hydroperoxy-trans-10, trans-12-octadecadienoic acid. Addition of a surfactant such as sodium dodecyl sulfate (SDS) or sodium cholate (SC) into the running buffer (20-30 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) or ammonium acetate, pH 9.5-11) was required to enhance the water solubility of the sample and selectivity of the separation. MEKC proved to be a promising new technique for the separation of the primary oxidation products of lipids giving results comparable to high performance liquid chromatography (HPLC). Partial separation of hydroperoxide isomers was also achieved using nonaqueous CE with methanol-acetonitrile-sodium cholate as running buffer.

Immunohistochemical and ELISA assays for biomarkers of oxidative stress in aging and disease.

Oxidative stress is apparent in pathology associated with aging and many age-related, chronic diseases, including atherosclerosis, diabetes mellitus, rheumatoid arthritis, and neurodegenerative diseases. Although it cannot be measured directly in biological systems, several biomarkers have been identified that provide a measure of oxidative damage to biomolecules. These include amino acid oxidation products (methionine sulfoxide, ortho-tyrosine (o-tyr) and dityrosine, chlorotyrosine and nitrotyrosine), as well as chemical modifications of protein following carbohydrate or lipid oxidation, such as N epsilon-(carboxymethyl)lysine and N epsilon-(carboxyethyl)lysine, and malondialdehyde and 4-hydroxynonenal adducts to amino acids. Other biomarkers include the amino acid cross-link pentosidine, the imidazolone adducts formed by reaction of 3-deoxyglucosone or methylglyoxal with arginine, and the imidazolium cross-links formed by the reaction of glyoxal and methylglyoxal with lysine residues in protein. These compounds have been measured in short-lived intracellular proteins, plasma proteins, long-lived extracellular proteins, and in urine, making them valuable tools for monitoring tissue-specific and systemic chemical and oxidative damage to proteins in biological systems. They are normally measured by sensitive high-performance liquid chromatography or gas chromatography-mass spectrometry methods, requiring both complex analytical instrumentation and derivatization procedures. However, sensitive immunohistochemical and ELISA assays are now available for many of these biomarkers. Immunochemical assays should facilitate studies on the role of oxidative stress in aging and chronic disease and simplify the evaluation of therapeutic approaches for limiting oxidative damage in tissues and treating pathologies associated with aging and disease. In this article we summarize recent data and conclusions based on immunohistochemical and ELISA assays, emphasizing the strengths and limitations of the techniques.

Peroxynitrite oxidises catechols to o-quinones

Nitration of phenolic compounds is a well-established mechanism on interaction with peroxynitrite. However, while nitration is the predominant reaction for monophenolic hydroxycinnamates, this does not take place with the catechol-containing hydroxycinnamate, caffeic acid. The aim of the present study was to investigate the mechanism of the chemical interaction of caffeic acid with peroxynitrite and to characterise the products formed. A novel compound was detected and characterised as the o-quinone of caffeic acid based on its reaction with nucleophilic thiol compounds, glutathione and l-cysteine. The same novel product was identified following the oxidation of caffeic acid in alkaline solutions confirming the identity of this species as a caffeic acid oxidation product.

Free and bound fatty acid oxidation products in archaeological ceramic vessels

While oxidation products of unsaturated fatty acids, for example dicarboxylic acids (hereafter diacids), must form during the use of unglazed ceramic vessels for the processing of animal and plant products, such components have never been observed during studies of absorbed lipids. Their absence from the extractable lipid fraction is presumed to be the result of their loss from potsherds through groundwater leaching. Lipid oxidation products including short-chain dicarboxylic acids, ω-hydroxy acids and longer-chain hydroxy and dihydroxy acids have now been observed as components probably covalently bound into solvent insoluble residues of potsherds recovered from waterlogged deposits. These components were only revealed following alkaline treatment of the insoluble residues. A similar mixture of diacids was observed in high abundance in the free lipid fraction of vessels recovered from an exceptionally arid deposit where groundwater leaching would never have occurred. These results confirm the formation of oxidation and probable polymerization products of unsaturated fatty acids during vessel use and burial.

Emulsification of crude oil by an alkane-oxidizingRhodococcusspecies isolated from seawater

A Rhodococcus species, the best of 99 oil-emulsifying bacteria isolated from globally distributed seawater samples, was characterized. The bacterium produced very stable oil-in-water emulsions from different crude oils with various contents of aliphatic and aromatic compounds by utilizing the C11to C33n-alkanes as carbon and energy sources. The presence of alkanes induced the formation of a hydrophobic cell surface that permitted oil-associated exponential growth and where an extensive emulsification of the residual oil and accumulation of acidic oxidation products occurred. The acidic products were consumed in a second step characterized by linear growth and an increasing number of cells growing in the water phase. Adhesion of cells resulted in some stabilization of oil droplets, but the most extensive emulsification occurred at the end of the exponential phase and coincided with an increasing number of cells in the water phase. No surfactant could be detected in the water phase during exponential growth, but a polymeric compound with emulsifying activity, tightly bound to the oil droplets, could be isolated. This suggests that the emulsification was caused by the release of the hydrophobic cell surface discarded by the cells during conditions of growth limitations.Key words: Rhodococcus,emulsification, adhesion, n-alkanes, hydrophobicity.

Restoration of catalytic activity beyond wild-type level in glucoamylase from Aspergillus awamori by oxidation of the Glu400-->Cys catalytic-base mutant to cysteinesulfinic acid.

Glucoamylase catalyzes the hydrolysis of glucosidic bonds with inversion of the anomeric configuration. Site-directed mutagenesis and three-dimensional structure determination of the glucoamylase from Aspergillus awamori previously identified Glu179 and Glu400 as the general acid and base catalyst, respectively. The average distance between the two carboxyl groups was measured to be 9.2 A, which is typical for inverting glycosyl hydrolases. In the present study, this distance was increased by replacing the catalytic base Glu400 with cysteine which was then oxidized to cysteinesulfinic acid. Initially, this oxidation occurred during attempts to carboxyalkylate the Cys400 residue with iodoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by high-pressure liquid chromatography in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on purified peptide fragments demonstrated that all enzyme derivatives contained the cysteinesulfinic acid oxidation product of Cys400. Subsequently, it was demonstrated that treatment of Glu400-->Cys glucoamylase with potassium iodide in the presence of bromine resulted in complete conversion to the cysteinesulfinic acid product. As expected, the catalytic base mutant Glu400-->Cys glucoamylase had very low activity, i.e., 0.2% compared to wild-type. The oxidation of Cys400 to cysteinesulfinic acid, however, restored activity (kcat) on alpha-1,4-linked substrates to levels up to 160% of the wild-type glucoamylase which corresponded to approximately a 700-fold increase in the kcat of the Glu400-->Cys mutant glucoamylase. Whereas Glu400-->Cys glucoamylase was much less thermostable and more sensitive to guanidinium chloride than the wild-type enzyme, the oxidation to cysteinesulfinic acid was accompanied by partial recovery of the stability.

Presence of dopa and amino acid hydroperoxides in proteins modified with advanced glycation end products (AGEs): amino acid oxidation products as a possible source of oxidative stress induced by AGE proteins.

Glycation and subsequent Maillard or browning reactions of glycated proteins, leading to the formation of advanced glycation end products (AGEs), are involved in the chemical modification of proteins during normal aging and have been implicated in the pathogenesis of diabetic complications. Oxidative conditions accelerate the browning of proteins by glucose, and AGE proteins also induce oxidative stress responses in cells bearing AGE receptors. These observations have led to the hypothesis that glycation-induced pathology results from a cycle of oxidative stress, increased chemical modification of proteins via the Maillard reaction, and further AGE-dependent oxidative stress. Here we show that the preparation of AGE-collagen by incubation with glucose under oxidative conditions in vitro leads not only to glycation and formation of the glycoxidation product Nepsilon-(carboxymethyl)lysine (CML), but also to the formation of amino acid oxidation products on protein, including m-tyrosine, dityrosine, dopa, and valine and leucine hydroperoxides. The formation of both CML and amino acid oxidation products was prevented by anaerobic, anti-oxidative conditions. Amino acid oxidation products were also formed when glycated collagen, prepared under anti-oxidative conditions, was allowed to incubate under aerobic conditions that led to the formation of CML. These experiments demonstrate that amino acid oxidation products are formed in proteins during glycoxidation reactions and suggest that reactive oxygen species formed by redox cycling of dopa or by the metal-catalysed decomposition of amino acid hydroperoxides, rather than by redox activity or reactive oxygen production by AGEs on protein, might contribute to the induction of oxidative stress by AGE proteins.

Characterization and classification of Tarfaya kerogen (South Morocco) based on its oxidation products

A 23-step alkaline potassium permanganate degradation of the kerogen from Moroccan Tarfaya oil shale was carried out, yielding oxidation products in 81.1 wt% yield. Detailed GC and GC-MS analyses of acid oxidation products and products of further controlled potassium permanganate degradation of high-molecular weight acids allowed quantitative estimation of the contribution of the various types of products and comparison with other kerogens studied following the same procedure. Oxidation products are dominantly aliphatic (56.6 wt% of total oxidation products) and aromatic (36.3 wt%) in confirmation of NMR results. Detailed analysis of each kind of aliphatic and aromatic acids indicates that the network is formed by aliphatic (rather short) cross-linked chains with inserted aromatic structures. The basic network is substantially substituted by aromatic moieties and alkyl chains acting as monosubstituents.

Fatty streak formation in fat-fed mice expressing human copper-zinc superoxide dismutase.

Studies in vitro have shown that copper-zinc superoxide dismutase (CuZn-SOD) inhibits a number of events putatively involved in atherogenesis, including cell-mediated oxidation of LDL. To investigate whether increased activity of CuZn-SOD reduces atherogenesis in vivo, we examined diet-induced fatty streak formation in CuZn-SOD transgenic mice (n = 24) as compared with their nontransgenic littermates (n = 28). Transgenic animals were originally created by introduction of an EcoRI-BamHI human genomic DNA fragment containing the CuZn-SOD gene and its regulatory elements into B6SJL zygotes. For the current studies, the transgene was bred for 12 generations into the atherosclerosis-susceptible C57BL/6 background. Animals were fed atherogenic diets (15% fat, 1.25% cholesterol, 0.5% Na cholate) starting at 100 weeks of age and extending for 18 weeks. At the end of the diet period, aortic SOD activity was two-fold higher in transgenics than nontransgenics (mean +/- SE: 46.7 +/- 5.8 versus 20.1 +/- 2.4 units/mg of protein, P < .001). Levels of protein-bound amino acid oxidation products (meta-, ortho-, and dityrosine) were either similar or lower in aorta and heart from transgenics as compared with nontransgenics, suggesting that amplification of CuZn-SOD activity above the normal complement had modest inhibitory effects on basal oxidative stress in these tissues. CuZn-SOD overexpression did not reduce the extent of lesion development as analyzed by quantitative lipid staining of serial sections of the proximal aorta; mean lesion areas (+/- SE) were 997 +/- 478 and 943 +/- 221 mu 2 in transgenics and nontransgenics, respectively. Notably, the range of values for lesion area was 2.2-fold greater in transgenics (0-8403 versus 0-3868 mu 2 in nontransgenics). Moreover, within this group, lesion area showed a significant positive correlation with SOD activity (r = .611, P < .03). These results do not support an antiatherogenic effect of Cu-Zn-SOD over expression and raise the possibility that high tissue SOD activity may potentiate atherogenesis in fat-fed atherosclerosis-susceptible mice [corrected].

Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes. Evidence against a generalized increase in oxidative stress in diabetes.

The glycoxidation products Nepsilon-(carboxymethyl)lysine and pentosidine increase in skin collagen with age and at an accelerated rate in diabetes. Their age-adjusted concentrations in skin collagen are correlated with the severity of diabetic complications. To determine the relative roles of increased glycation and/or oxidation in the accelerated formation of glycoxidation products in diabetes, we measured levels of amino acid oxidation products, distinct from glycoxidative modifications of amino acids, as independent indicators of oxidative stress and damage to collagen in aging and diabetes. We show that ortho-tyrosine and methionine sulfoxide are formed in concert with Nepsilon-(carboxymethyl)lysine and pentosidine during glycoxidation of collagen in vitro, and that they also increase with age in human skin collagen. The age-adjusted levels of these oxidized amino acids in collagen was the same in diabetic and nondiabetic subjects, arguing that diabetes per se does not cause an increase in oxidative stress or damage to extracellular matrix proteins. These results provide evidence for an age-dependent increase in oxidative damage to collagen and support previous conclusions that the increase in glycoxidation products in skin collagen in diabetes can be explained by the increase in glycemia alone, without invoking a generalized, diabetes-dependent increase in oxidative stress.

Arachidonic acid and phospholipids oxidation products as the regulators of protein kinase c activity in vitro: E.L.Mal'tseva Inst. Chem. Physics RAS, Moscow, (Russia)

Arachidonic acid and phospholipids oxidation products as the regulators of protein kinase c activity in vitro: E.L.Mal'tseva Inst. Chem. Physics RAS, Moscow, (Russia)

Efficient scavenging of fatty acid oxidation products by aminoguanidine.

Lipid oxidation leads to the formation of reactive aldehydes that may play an important role in atherogenesis by altering the normal pathway of lipoprotein metabolism and by exerting toxic effects on vascular wall components. Recent studies indicate that advanced glycation end products, which form spontaneously from the reaction of reducing sugars with amino groups, may promote oxidative damage in vivo. Moreover, the pharmacological inhibitor of advanced glycation aminoguanidine has been shown to lower circulating low-density lipoprotein levels in human subjects and to inhibit certain oxidative reactions in vitro. To define more precisely the potential interaction of AG with oxidized lipids, we have studied and identified the major products that form from the reaction of AG with the oxidation products 4-hydroxynonenal and malondialdehyde. AG was found to be an efficient scavenger of alpha,beta-unsaturated aldehydes when compared to nucleophilic amino acids (Cys, Lys, His), suggesting that one of its mechanisms of action in vivo is to protect tissue constituents from the damaging effects of oxidative stress.