Free radical oxidation of lipids and thiol groups in the formation of a cataract

Content of primary (diene conjugates), secondary (ketodienes), end (Schiff's bases) products of free radical oxidation (FRO) of lipids was determined, as well as content of total and non-protein-bound thiols in human lens at different stages of cataractogenesis. Lens opacity was estimated by quantitative morphometric analysis. Participation of FRO of lipids in lens opacity is proved. It is shown that total thiols of lens fibres are rapidly inactivated when the intensity of lipid FRO is increased at the expense of the fall of glutathione level. These processes promote the formation of high molecular protein aggregates in the lens and cataract development. Coefficients of linear correlation between the indicated parameters are presented. A conclusion is drawn concerning possible prevention of cataract development by decreasing the level of accumulation of lipid peroxides and by maintaining high concentration of reduced glutathione in the lens.     

Free radical oxidation of lipids in the rat hypothalamus under stress after cortisol pretreatment

Free radical oxidation of lipids in the rat hypothalamus after cortisol pretreatment weas investigated. Three-fold cortisol injection (25 mg/kg, once per day) resulted in significant decrease of average level of Schiffbases, the final products of lipid oxidation. Stress causes increase of average level of Schiff bases in the control animals (without cortisol pretreatment). Cortisol pretreatment potentiated stress-induced changes in the processes of free radical oxidation of lipids in the rats hypothalamus.     

Free radical oxidation (autoxidation) of alkenones and other lipids in cells of Emiliania huxleyi

Cells of the coccolithophorid Emiliania huxleyi strain CS-57 grown under an atmosphere of air+0.5% CO(2) showed oxidative damage after 10 days growth with concomitant and major changes to the lipid composition. The fatty acid profile was strongly altered and lacked appreciable amounts of the polyunsaturated fatty acids (PUFA: C(18:5), C(18:3) and C(22:6)) typical of healthy cells. Oxidation products of these PUFA could not be detected, but monounsaturated fatty acids proved to be good indicators of oxidative processes. The presence (after NaBH(4)-reduction) of a high proportion of 11-hydroxyoctadec-cis-9-enoic and 8-hydroxyoctadec-cis-9-enoic acids showed that the degradation of oleic acid involved mainly free radical oxidation processes (70-75% autoxidation and 20-25% photooxidation). We also detected large amounts of degradation products of the oxidation product 9,10-epoxyoctadecanoic acid including diols, methoxyhydrins and chlorohydrins. These oxidative effects were found in all the lipid classes examined. Products included significant amounts of chlorophyll side-chain autooxidation products Z- and E-3,7,11,15-tetramethylhexadec-3-ene-1,2-diols and Z-and E-3,7,11,15-tetramethylhexadec-2-ene-1,4-diols, while phytyldiol was present in relatively low proportions. Delta(5)-3beta,7-epimeric unsaturated steroidal diols arising from the autooxidation of the Delta(5) double bond of epi-brassicasterol and minor amounts of Delta(4)-3beta,6-diols were also detected. Long-chain unsaturated ketone (alkenone) content per cell was much higher in the presence of 0.5% CO(2) likely due to carbon storage under these conditions. The proportions of di- and tri-unsaturated alkenones was relatively stable throughout the growth cycle in the absence of additional CO(2), but not when grown with 0.5% CO(2). The detection of characteristic alkenone autoxidation products in cells grown under these latter conditions allowed us to attribute the significant increase in index observed to the involvement of free radical oxidation processes.     

Phosphate radical induced oxidation of pyrimidine bases in aqueous solution

The photooxidation of pyrimidine bases viz., uracil and cytosine in presence of peroxydiphosphate (PDP) in aqueous solution at natural pH (approximately 7.5) has been carried out in a quantum yield reactor using a high-pressure mercury lamp. The rates of oxidation and quantum yields of pyrimidine oxidation have been found to increase with increase in [PDP] while they are independent of [pyrimidine] and light intensity. On the basis of these experimental results, product analysis and existence of isosbestic points a probable mechanism is suggested in which peroxydiphosphate ion on photolysis gives phosphate radical anions which initiates the reaction by adding to C(5) or C(6) of pyrimidine base leading to the formation of pyrimidine radical via radical cation or hydrolysis. This further reacts with PDP and gives the final products 5,6-dihydroxy pyrimidine and isobarbituric acid.     

A novel lecithin-cholesterol acyltransferase antioxidant activity prevents the formation of oxidized lipids during lipoprotein oxidation.

Recent investigations suggest that high-density lipoprotein (HDL) may play an anti-atherogenic role as an antioxidant and inhibit the oxidative modification of low-density lipoprotein (LDL). The antioxidant activity of HDL has been proposed to be associated with several HDL-bound proteins. We have purified one HDL-associated protein, lecithin:cholesterol acyltransferase (LCAT), to apparent homogeneity and have found that LCAT is not only capable of esterifying cholesterol in the plasma, but can also prevent the accumulation of oxidized lipids in LDL. Addition of pure human LCAT to LDL or palmitoyl-linoleoyl phosphatidylcholine/sodium cholate (PLPC) micelles inhibits the oxidation-dependent accumulation of both conjugated dienes and lipid hydroperoxides. LCAT also inhibits the increase of net negative charge that occurs during oxidation of LDL. LCAT has the ability to prevent spontaneous oxidation and Cu2+ and soybean lipoxygenase-catalyzed oxidation of lipids. The antioxidant activity of LCAT appears to be enzymatic, since the enzyme is active for up to 10 h in the presence of mild free-radical generators. The catalytic serine, residue 181, may mediate this activity and act as a reusable proton donor. Chemical modification of the active serine residue with diisopropylfluorophosphate completely inhibits the ability of LCAT to prevent lipid oxidation. Thus, in addition to its well-characterized phospholipase and acyltransferase activities, LCAT can also act as an antioxidant and prevent the accumulation of oxidized lipid in plasma lipoproteins.     

Induction of DNA strand breakage and base oxidation by nitroxyl anion through hydroxyl radical production.

Nitroxyl anion (NO-), the one-electron reduction product of nitric oxide (NO*), has been reported to be formed under various physiological conditions and to be cytotoxic, although the mechanism responsible for the toxic effects has not been identified. We have studied the effects of NO- generated from Angeli's salt (sodium trioxodinitrate) or Piloty's acid (N-hydoxybenzenesulfonamide) on DNA strand breakage and DNA base oxidation in vitro. Induction of strand breakage was dose- and time-dependent upon incubation of plasmid pBR322 with Angeli's salt or Piloty's acid. Similarly, 8-oxo-2'-deoxyguanosine and malondialdehyde were formed when calf-thymus DNA or 2'-deoxyribose, respectively, were incubated with Angeli's salt. Electron acceptors (ferricyanide, 4-hydroxy-TEMPO), that convert NO to NO*, inhibited the reactions, indicating that NO , but not NO*, is responsible for the reactions. Furthermore, the reactions were also inhibited by the presence of hydroxyl radical (HO*) scavengers, antioxidants, metal chelators and superoxide dismutase and catalase, implying involvement of free HO*. These results suggest that NO- is a possible endogenous source of HO*, that may be formed either directly from the reaction product of NO- with NO* (N2O2*-) or indirectly through H2O2 formation. Thus NO may play an important role as a cause of diverse pathophysiological conditions such as inflammation and neurodegenerative diseases.     

Cytochrome P450cam-catalyzed oxidation of a hypersensitive radical probe.

trans-1-Phenyl-2-vinylcyclopropane, a hypersensitive radical probe, is oxidized by cytochrome P450cam (CYP101) to a diastereomeric mixture of the corresponding epoxide (81%), (trans-2-phenylcyclopropyl)acetaldehyde (6%), and trans-5-phenyl-2-penten-1,5-diol (13%). trans-5-Phenyl-2-penten-1-ol and (trans-2-phenylcyclopropyl)ethane-1,2-diol are not detectably formed. Authentic standards of all the products have been synthesized and used to establish the identities (or the absence) of the metabolites. Studies with [18O]H2O demonstrate that the oxygens at positions 1 and 5 in the rearranged diol derive from molecular oxygen and water, respectively. Catalytic turnover of the enzyme is required for product formation from the olefin, but incubation of the epoxide metabolite with the enzyme, or with buffer alone, yields both the aldehyde and the rearranged diol products. The absence of trans-5-phenyl-2-penten-1-ol implies that the lifetime of the putative radical intermediate is so short that its existence as a discrete entity is questionable. A cationic intermediate is unlikely but cannot be excluded because the same metabolites are formed in a secondary reaction, even at pH 8.0, from the epoxide. The results provide no evidence for the involvement of radicals or cations in the epoxidation reaction, in agreement with results on the oxidation of olefins in organic solvents by metalloporphyrin catalysts.     

Yolk lipids.

The mature egg yolk of the domestic hen possesses remarkably constant lipid and lipoprotein composition despite much variation in dietary and environmental conditions. The greatest differences are seen in the fatty acid composition of the triacylglycerols which may show significant alterations in the content of the minor acids including certain polyunsaturated acids. The lipid class composition appears to be minimally affected by dietary influences, including the cholesterol content of the diet. The limited dietary influence on the yolk lipid composition extends to different strains of the hens. Genetic selection has led to some increase in the cholesterol content of the egg, but the desired lowering of the cholesterol content of egg yolk has not been realized. Likewise, production of a polyunsaturated fatty acid egg does not appear to be practical. As a result the egg yolk continues to provide a food product of nearly constant composition, which serves to maintain its chemical and physico-chemical properties for reliable utilization in the baking, cosmetic and pharmaceutical industries. The great uniformity in the composition of the egg yolk phospholipids makes them desirable starting materials for partial chemical resynthesis of glycerophospholipids. Partial hydrogenation of the egg yolk lipids promises to further increase the utility of the product as a desirable material for the manufacture of liposomes and liposome based drug products. In contrast, the constancy of the egg yolk composition and the inability to alter it significantly by dietary or genetic means also renders egg yolk undesirable for unlimited human consumption. Excessive ingestion of egg yolk raises plasma lipid and cholesterol levels which are believed to contribute to the development of heart disease. The physico-chemical and biological properties of egg yolk apoproteins have been less extensively investigated and their function is less well understood. The finding that phosvitin is a effective chelator of metal ions and thus an effective antioxidant demonstrates that egg yolk lipoproteins possess as yet unexplored potential for beneficial nutritional, medical and industrial application.     

A carbon-centered free radical intermediate in the prostaglandin synthetase oxidation of arachidonic acid. Spin trapping and oxygen uptake studies

The ESR spin-trapping technique has been used to identify a free radical involved in the oxygenation of arachidonic acid by ram seminal vesicle microsomes. The ESR spectrum of the radical adduct indicates that a carbon-centered arachidonic acid free radical has been observed. The formation of this species is inhibited by indomethacin, but not by phenol, and it is probably the first intermediate formed during the prostaglandin synthetase-catalyzed oxidation of arachidonic acid. The chemical identity of the trapped radical was substantiated with an independent synthesis of a closely related radical adduct.     

Time-course of oxidation of lipids in human cerebrospinal fluid in vitro

Oxidative mechanisms play an important role in the pathogenesis of Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases. To assess whether the oxidation of brain lipoproteins plays a role in the development of these pathologies, we investigated whether the lipoproteins of human cerebrospinal fluid (CSF) are susceptible to oxidative modification in vitro. We studied oxidation time-course for up to 100 h of human CSF in the absence (autooxidation) or presence of exogenous oxidants. Autooxidation of diluted CSF was found to result in a slow accumulation of lipid peroxidation products. The time-course of lipid hydroperoxide accumulation revealed three consecutive phases, lag-phase, propagation phase and plateau phase. Qualitatively similar time-course has been typically found in human plasma and plasma lipoproteins. Autooxidation of CSF was accelerated by adding exogenous oxidants, delayed by adding antioxidants and completely inhibited by adding a chelator of transition metal ions. Autooxidation of CSF also resulted in the consumption of endogenous ascorbate, alpha-tocopherol, urate and linoleic and arachidonic acids. Taking into account that (i) lipid peroxidation products measured in our study are known to be derived from fatty acids, and (ii) lipophilic antioxidants and fatty acids present in CSF are likely to be located in CSF lipoproteins, we conclude that lipoproteins of human CSF are modified in vitro during its autooxidation. This autooxidation appears to be catalyzed by transition metal ions, such as Cu(II) and Fe(III), which are present in native CSF. These data suggest that the oxidation of CSF lipoproteins might occur in vivo and play a role in the pathogenesis of neurodegenerative diseases.     

A functional state of the sphingomyelin cycle and activity of free radical oxidation of rat liver lipids at different phases of starvation

A functional state of the sphingomyeline cycle and its links with processes of free radical lipid oxidation have been investigated in rat liver during starvation of animals without any restriction of access to drinking water at day 1, 2, 3 (phase I) and day 6 (phase II of starvation). The maximal values of the ceramide/sphingomyelin ratio and activities of neutral sphingomyelinase and executive caspase-3 were found in rat livers at day 3 of starvation. Since day 3 of starvation an increase of tumour necrosis factor-α, one of neutral sphingomyelinase activators, was detected in serum. During the major part of phase I of starvation the intensity of liver free radical lipid peroxidation was comparable to that of control due to competent functioning of the antioxidant defense system. Transition of phase I to phase II of starvation (day 6 of the experiment) was accompanied by the development of oxidative stress associated with depletion of the antioxidant defense system. The results obtained in this study suggest that phase I of starvation favors realization of the ceramide-mediated proapoptotic signaling in the liver. In our viewpoint, ceramide-mediated apoptosis is one of mechanisms used optimization of liver cellular population within the frame of metabolic adaptation. In rat liver phase I of starvation was characterized by prevailing of the ceramide-mediated proapoptotic signaling, while in phase II oxidative stress dominated.     

The enzymatic oxidation of Desferal to a nitroxide free radical

Desferrioxamine mesylate (Desferal), a transition metal ion chelator, has been used to inhibit the in vitro redox cycling of transition metal ions. ESR spectroscopy was utilized to detect and identify Desferal's one-electron oxidation product. We demonstrate that a horseradish peroxidase/H₂O₂ system, a xanthine oxidase/hypoxanthine system, and a hydroxyl radical-generating system are all capable of oxidizing Desferal to a nitroxide free radical. The same 9-line ESR spectrum (g = 2.0065, a^N = 7.85 G, a^H(2) = 6.35 G) was detected in all of the above systems. We, therefore, stress that care must be taken when using Desferal as a transition metal ion chelator to keep its concentration low enough to minimize these reactions, or to use a different metal ion chelator.     

Free radical oxidation of proteins and its relationship with functional state of organisms

Most reactive oxygen species (ROS) in living organisms are produced as byproducts of many processes. Being highly active, ROS interact with virtually all cellular components particularly modifying their properties. In this review, detailed analysis of chemical modifications of proteins on their interaction with ROS is given with particular interest in cleavage of polypeptide chains and oxidation of side chains of amino acid residues. Special attention has been paid to identification of products of free radical modification of proteins with a focus on the formation of additional carbonyl groups, which are the most frequently used markers of these processes. Functional consequences of protein modification by ROS depend on the nature of ROS and protein as well as particular conditions of their interaction. The relationship between protein oxidation and functional state of organisms, particularly aging, hyperoxia and hypoxia, and heat shock, as well as with different pathologies has been analyzed. The final part of the article is devoted to possible ways of protecting proteins against oxidation in vivo. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 8, pp. 995–1017.     

Uric acid oxidation by peroxynitrite: multiple reactions, free radical formation, and amplification of lipid oxidation

Uric acid has been considered to be an efficient scavenger of peroxynitrite but the reaction between urate and peroxynitrite has been only partially characterized. Also, previous studies have indicated that urate may increase peroxynitrite-mediated oxidation of low density lipoprotein (LDL). Here, we examined the reaction between urate and peroxynitrite by combining kinetic, oxygen consumption, spin trapping, and product identification studies; in parallel, we tested the effect of urate upon peroxynitrite-mediated lipid oxidation. Our results demonstrated that urate reacts with peroxynitrite with an apparent second order rate constant of 4.8 x 10(2) M(-1). s(-1) in a complex process, which is accompanied by oxygen consumption and formation of allantoin, alloxan, and urate-derived radicals. The main radical was identified as the aminocarbonyl radical by the electrospray mass spectra of its 5, 5-dimethyl-l-pyrroline N-oxide adduct. Mechanistic studies suggested that urate reacts with peroxynitrous acid and with the radicals generated from its decomposition to form products that can further react with peroxynitrite anion. These many reactions may explain the reported efficiency of urate in inhibiting some peroxynitrite-mediated processes. Production of the aminocarbonyl radical, however, may propagate oxidative reactions. We demonstrated that this radical is likely to be the species responsible for the effects of urate in amplifying peroxynitrite-mediated oxidation of liposomes and LDL, which was monitored by the formation of lipid peroxides and thiobarbituric acid-reactive substances. The aminocarbonyl radical was not detectable during urate attack by other oxidants and consequently it is unlikely to be responsible for all previously described prooxidant effects of uric acid.     

Time-course of oxidation of lipids in human cerebrospinal fluid in vitro

Oxidative mechanisms play an important role in the pathogenesis of Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases. To assess whether the oxidation of brain lipoproteins plays a role in the development of these pathologies, we investigated whether the lipoproteins of human cerebrospinal fluid (CSF) are susceptible to oxidative modification in vitro. We studied oxidation time-course for up to 100 h of human CSF in the absence (autooxidation) or presence of exogenous oxidants. Autooxidation of diluted CSF was found to result in a slow accumulation of lipid peroxidation products. The time-course of lipid hydroperoxide accumulation revealed three consecutive phases, lag-phase, propagation phase and plateau phase. Qualitatively similar time-course has been typically found in human plasma and plasma lipoproteins. Autooxidation of CSF was accelerated by adding exogenous oxidants, delayed by adding antioxidants and completely inhibited by adding a chelator of transition metal ions. Autooxidation of CSF also resulted in the consumption of endogenous ascorbate, α-tocopherol, urate and linoleic and arachidonic acids. Taking into account that (i) lipid peroxidation products measured in our study are known to be derived from fatty acids, and (ii) lipophilic antioxidants and fatty acids present in CSF are likely to be located in CSF lipoproteins, we conclude that lipoproteins of human CSF are modified in vitro during its autooxidation. This autooxidation appears to be catalyzed by transition metal ions, such as Cu(II) and Fe(III), which are present in native CSF. These data suggest that the oxidation of CSF lipoproteins might occur in vivo and play a role in the pathogenesis of neurodegenerative diseases.