Oxidative Damage and the Preservation of Organic Artefacts

Organic artefacts degrade by a number of mechanisms. Autoxidation is one of the most important and involves free radicals. The importance of free radical reactions in the degradation of paper and other cellulosic materials, rubber and other organics is described. In the case of paper, treatment with calcium and magnesium compounds can inhibit oxidation, while transition metals increase the rate. Antioxidants have not been used a great deal due to unwanted side effects. The Russell effect, a technique for detecting oxidation using photographic materials. is described.     

A modified assay system for enzymes involved in N-acetyl group transfer reactions: its use to study enzymes involved in ornithine biosynthesis in plants

An enzymatic method for the determination of the amount of free fatty acids released from triglyceride by lipoprotein lipase is described. The quantity of free fatty acids present in media before and after incubation is measured spectrophotometrically by the oxidation of NADH in the final reaction of a series of coupled enzymatic reactions. This assay for lipoprotein lipase is unlike previously described assays in that radioactive substrates or titration procedures are not used in the free fatty acid determination. In addition, another method for assay of lipoprotein lipase activity that involves the separation of free fatty acids from triglycerides by adsorption chromatography with Florisil as a stationary phase is described.     

The importance of inhibiting polyphenol oxidase in the extraction of fraction 1 leaf protein

The interaction between polyphenol oxidation products and Fraction 1 protein produces a modified protein of increased electrophoretic mobility. A method is described for the extraction of red clover leaf Fraction 1 protein free of polyphenol oxidation products by inhibiting polyphenol oxidase with sodium diethyldithiocarbamate.     

Main lipophilic extractives in different paper pulp types can be removed using the laccase–mediator system

Lipophilic extractives in wood and other lignocellulosic materials exert a negative impact in pulp and paper manufacturing causing the so-called pitch problems. In this work, the appropriateness of an enzymatic treatment using the laccase–mediator system for pitch biocontrol is evaluated. With this purpose, three pulp types representative for different raw materials and pulping processes—eucalypt kraft pulping, spruce thermomechanical pulping, and flax soda-anthraquinone pulping—were treated with a high-redox-potential laccase from the basidiomycete Pycnoporus cinnabarinus in the presence of 1-hydroxybenzotriazole as a redox mediator. The gas chromatography and gas chromatography/mass spectrometry analyses of the lipophilic extractives from the enzymatically treated pulps revealed that the laccase–mediator treatment completely or greatly removed most of the pitch-causing lipophilic compounds present in the different pulps including: (1) free and conjugated sitosterol in eucalypt paper pulp; (2) resin acids, sterol esters, and triglycerides in spruce pulp; and (3) sterols and fatty alcohols in the flax pulp. Different amounts of free and conjugated 7-oxosterols were found as intermediate products in the oxidation of pulp sterols. Therefore, the laccase–mediator treatment is reported as an efficient method for removing pitch-causing lipophilic compounds from paper pulps obtained from hardwood, softwood, and nonwoody plants.     

Recent developments in photochemical and chemical AOPs in water treatment: a mini-review

Photochemical and chemical oxidation methods, belonging to a wide group of advanced oxidation processes (AOPs), are extensively studied but still under constant development. The aim of this study was to review the recent developments mainly in UV/H₂O₂ process and Fenton’s reactions. Ultraviolet (UV) radiation is an essential factor in photochemical oxidation. In the development of UV radiation sources the energy efficiency and the use of toxicant free materials have become major objectives. Also the emitted wavelength plays a significant role. Besides, there are alternatives for UV radiation sources, such as radio frequency (RF) plasmas, which can operate in water without additional chemicals. A remarkable development in the field of chemical oxidation has been novel catalyst materials for Fenton’s reaction enabling the efficient reaction in neutral or almost neutral conditions, instead of pH below 3. In addition, varieties of method integrations, e.g., combinations of AOPs or AOPs integrated to biological processes, and other enhancements such as combining ultrasound (US) with AOP have led to improved water treatment efficiencies.     

An NADH-linked luciferase assay for glycogen: the preparation of glycogen-free, viable human eccrine sweat glands

A glycogen assay based on bacterial NADH luciferase is described. It is free of tissue interference. The detection limit is 0.12 nmol glycogen, and the coefficient of variation is 5.5%. A method of depleting human eccrine sweat glands while retaining their viability is described. This depends on their incubation in 10(-5) M acetylcholine and 1 mM pyruvate. This method may be applicable to other tissues. The evidence for the viability of glycogen-depleted human eccrine sweat glands is reported and includes tissue contents of ATP and the rates of oxidation of glucose, pyruvate, beta-hydroxybutyrate, and palmitate.     

Fixed phagocytes in the freshwater snail Lymnaea stagnalis

Morphological and histochemical examination of the blood and connective tissue of the freshwater snail Lymnaea stagnalis injected with various types of foreign particulate materials has shown the existence of free as well as fixed phagocytic cells. The morphology of the fixed phagocytes is described, and the phagocytic system of the snail is compared with that of other molluscan species.     

Alternate method of calculating the free-energy change accompanying the growth of saccharomyces cerevisiae (Hansen) on three substrates.

A method is described for determining the free energy of formation of the cells of Saccharomyces cerevisiae (Hansen) that are formed as the result of anaerobic growth on glucose, and aerobic growth on glucose and ethanol. The method is based on the direct relationship that exists between the enthalpy changes and the free-energy changes that accompany the oxidation of 1 g cellular material formed during these growth reactions and the degree of reduction of the same material. When the results of these calculations are used together with the free energies of formation of the reactants and of other products of a given growth reaction, it becomes possible to calculate the free-energy change accompanying this reaction. These free-energy changes are in excellent agreement with those calculated by another method based on the hypothesis that the free-energy change accompanying the conversion of the substrate plus other reactants into cellular material plus other products is equal to zero.     

Free radical oxidation of cardiolipin: chemical mechanisms, detection and implication in apoptosis, mitochondrial dysfunction and human diseases

Cardiolipin (CL) is a mitochondria-specific phospholipid and is critical for maintaining the integrity of mitochondrial membrane and mitochondrial function. CL also plays an active role in mitochondria-dependent apoptosis by interacting with cytochrome c (cyt c), tBid and other important Bcl-2 proteins. The unique structure of CL with four linoleic acid side chains in the same molecule and its cellular location make it extremely susceptible to free radical oxidation by reactive oxygen species including free radicals derived from peroxidase activity of cyt c/CL complex, singlet oxygen and hydroxyl radical. The free radical oxidation products of CL have been emerged as important mediators in apoptosis. In this review, we summarize the free radical chemical mechanisms that lead to CL oxidation, recent development in detection of oxidation products of CL by mass spectrometry and the implication of CL oxidation in mitochondria-mediated apoptosis, mitochondrial dysfunction and human diseases.     

Mechanism(s) for the metabolism of mitoxantrone: electron spin resonance and electrochemical studies

Mitoxantrone has been reported to lack certain properties that characterize quinone containing antitumor agents that undergo enzymatic reduction. These properties are the stimulation of NADPH oxidation, the stimulation of oxygen consumption by microsomes and reductases and, the absence of oxygen free radicals during these reactions. Having these properties implies the presence of a futile redox cycle that requires the generation and the oxidation of a semiquinone free radical. It would follow that if mitoxantrone does not redox cycle in the presence of reductases, then the semiquinone free radical is not produced or, if it is formed, it reacts quickly to form diamagnetic products. However, using liver microsomes, there are reports of the formation of the mitoxantrone free radial anion. In this paper we investigated the mitoxantrone free radical anion generated electrochemically and found that in the presence of oxygen it behaved like other semiquinones. That is, it is oxidized to the parent compound (presumably generating oxygen free radicals), indicating the ability to redox cycle. The reduction potential to generate such free radical in aqueous medium is very high (-0.79 V) when compared to diaziquone (-0.36 V) and Adriamycin (-0.6 V). This suggests that mitoxantrone may not be a substrate for reductases. Under reductive conditions with purified NADPH cytochrome P-450 reductase which very easily reduces diaziquone and Adriamycin, mitoxantrone was not reduced. However, under the same conditions, mitoxantrone was oxidized by the prototype oxidase horseradish peroxidase with the production of a mitoxantrone free radical. This oxidation was accompanied by a drastic change in color and the formation of a dark precipitate. Because microsomes contain a variety of enzymes, we suggest that the previously observed free radical in microsomes is probably due to the oxidation of mitoxantrone. In this theory, this product is probably a polymer which would not require oxygen to be formed. Thus, under oxidative conditions, the mitoxantrone free radical cation will also display impaired redox activity.     

Palmitate oxidation by the mitochondria from volume-overloaded rat hearts

In this work, an attempt was made to identify the reasons of impaired long-chain fatty acid utilization that waspreviously described in volume-overloaded rat hearts. The most significant data are the following: (1) The slowing down of long-chain fatty acid oxidation in severely hypertrophied hearts cannot be related to a feedback inhibition of carnitine palmitoyltransferase I from an excessive stimulation of glucose oxidation since, because of decreased tissue levels of L-carnitine, glucose oxidation also declines in volume-overloaded hearts. (2) While, in control hearts, the estimated intracellular concentrations of free carnitine are in the range of the respective K_m of mitochondrial CPT I, a kinetic limitation of this enzyme could occur in hypertrophied hearts due to a 40% decrease in free carnitine. (3) However, the impaired palmitate oxidation persists upon the isolation of the mitochondria from these hearts even in presence of saturating concentrations of L-carnitine. In contrast, the rates of the conversion of both palmitoyl-CoA and palmitoylcarnitine into acetyl-CoA are unchanged. (4) The kinetic analyses of palmitoyl-CoA synthase and carnitine palmitoyltransferase I reactions do not reveal any differences between the two mitochondrial populations studied. On the other hand, the conversion of palmitate into palmitoylcarnitine proves to be substrate inhibited already at physiological concentrations of exogenous palmitate. The data presented in this work demonstrate that, during the development of a severe cardiac hypertrophy, a fragilization of the mitochondrial outer membrane may occur. The functional integrity of this membrane seems to be further deteriorated by increasing concentrations of free fatty acids which gives rise to an impaired functional cooperation between palmitoyl-CoA synthase and carnitine palmitoyltransferase I. In intact myocardium, the utilization of the generated in situ palmitoyl-CoA can be further slowed down by decreased intracellular concentrations of free carnitine.     

Methionine sulphoxide reductases revisited: free methionine as a primary target of H2O2 stress in auxotrophic fission yeast

Amino acid methionine can suffer reversible oxidation to sulphoxide and further irreversible over‐oxidation to methionine sulphone. As part of the cellular antioxidant scavenging activities are the methionine sulphoxide reductases (Msrs), with a reported role in methionine sulphoxide reduction, both free and in proteins. Three families of Msrs have been described, but the fission yeast genome only includes one representative for two of these families: MsrA/Mxr1 and MsrB/Mxr2. We have investigated their role in methionine reduction and H₂O₂ sensitivity. We show here that MsrA/Mxr1 is able to reduce free oxidized methionine. Cells lacking each one of the genes are not significantly sensitive to different types of oxidative stresses, neither display altered life span. However, only when deletion of msrA/mxr1 is combined with deletion of met6, which confers methionine auxotrophy, the survival upon H₂O₂ stress decreases by 100‐fold. In fact, cells lacking only Met6, and which therefore require addition of methionine to the growth media, are extremely sensitive to H₂O₂ stress. These and other evidences suggest that oxidation of free methionine is a primary target of peroxide toxicity in cells devoid of methionine biosynthetic capacity, and that an important role of Msrs is to recycle this oxidized free amino acid.     

Preparation of metal ion buffers for biological experimentation: a methods approach with emphasis on iron and zinc

Transition metal ions are a challenge to study in physiology because of problems associated with solubility, oxidation, binding, and attaining appropriate free activities in solution. This review discusses these problems and potential ways of accommodating them. Special attention is given to iron and zinc ions, but many of the concepts can be applied for studying other transition metals. Selection of reagents appropriate for metal work (including water, salts, noncomplexing pH buffers) is briefly discussed. Calculation of the solubility product (K(sp)) for common iron and zinc precipitates is covered, as well as techniques used to solubilize Fe(3+) with organic chelates. Factors that affect Fe(2+) oxidation are mentioned, and the use of ascorbate as a reducing agent is considered. Measurement of the rate of Fe(2+) oxidation (or Fe(3+) reduction) with the Fe(2+) chromophores ferrozine and BPS is also discussed. Generation of a free metal ion activity through use of metal buffers (chelators) is discussed. Theoretical problems associated with this technique are explored, and selected shareware metal ion buffer calculators are described. Finally, techniques for measuring and minimizing nonspecific binding of iron and zinc ions to biological membranes are considered.     

Liquid chromatographic analysis of sebum lipids and other lipids of medical interest

A technique is described for the high-pressure liquid chromatographic (HPLC) analysis of sebum lipid classes. The lipid classes present in sebum are separated by gradient elution HPLC from a microparticulate silica column and detected using a moving-wire detector. The system described can be linked to a computer. Quantitation can be carried out by comparing peak areas obtained with those of an internal standard. Peak trapping for further investigations of the separated components, for example by gas chromatography—mass spectrometry, is very easy.Sebum lipids are separated into the following lipid classes: hydrocarbons and squalene, cholesterol esters and wax esters, fatty acids as their methyl esters, triglycerides, 1,3-diglycerides, 1,2-diglycerides, free cholesterol, monoglycerides and other polar materials. Besides to sebum, the method has been successfully applied to other lipid mixtures, such as serum lipids. Examples of other applications are shown.     

Sterilization by electrohydraulic discharges

High voltage electric discharges between two electrodes immersed in a liquid (“electrohydraulic discharges”) inactivate microorganisms suspended in the liquid. The intense pulse of UV radiation emitted from the plasma formed between the electrodes causes most of the bactericidal effects, rather than shock waves, or free radicals or other chemical species formed in the liquid medium. A method of sterilizing materials without contamination from electrode debris is described. Possible applications and limitations of the technique are outlined.     

Terminal oxidation and the effects of zinc in prostate versus liver mitochondria

Although the total zinc content of cells generally approximates 0.2 mM, the cytosolic free zinc ion concentration is negligible (subnanomolar concentrations). However, all reported studies of effects of zinc on cellular respiration and terminal oxidation involved microM-mM levels of free zinc ions. Prostate cells and their mitochondria accumulate 3-10 fold more zinc than other mammalian cells. We considered that a cytosolic pool of mobile reactive low molecular weight zinc ligands could inhibit respiration and terminal oxidation. The effects of ZnLigands, especially ZnCitrate, versus free Zn++ ions on respiration and terminal oxidation were studied with prostate and liver mitochondria. ZnLigands were equally as effective as free Zn++ ions in the inhibition of respiration and terminal oxidation of both prostate and liver mitochondria, which supports our concept that zinc can be transferred from cytosolic donor ZnLigands directly to zinc-binding sites of terminal oxidation components. Also, the respiration and specific activities of terminal oxidation components of prostate mitochondria are 20-50% of liver mitochondria. Zinc inhibition and inherently low levels of electron transport components are likely major factors responsible for the low respiration that characterizes prostate cells.     

Effect of glucose ingestion on the metabolism of free fatty acids in human subjects

The rate of appearance of (14)CO(2) in expired air after the injection of a single dose of NaH(14)CO(3) has been determined in normal individuals both in the fasted and fed states. These data were combined with previously obtained results on the rate of disappearance of injected palmitate-(14)C from the bloodstream, to give a multicompartmental analysis of free fatty acid oxidation and esterification. The results confirm that glucose feeding promptly inhibits the rate of free fatty acid oxidation to CO(2). The "irreversible disposal rate," or irreversible flux of free fatty acids from the plasma, was also consistently reduced by glucose feeding. The diminution in irreversible disposal, not accounted for entirely by reduction of direct oxidation, must indicate suppression of other disposal mechanisms, including net esterification of free fatty acids. An average drop of 49% in "net esterification" when glucose was given may be compared with the 65% inhibition of rapid free fatty acid oxidation.     

Routes of formation and toxic consequences of lipid oxidation products in foods.

Lipid oxidation in foods is initiated by free radical and/or singlet oxygen mechanisms which generate a series of autocatalytic free radical reactions. These autoxidation reactions lead to the breakdown of lipid and to the formation of a wide array of oxidation products. The nature and proportion of these products can vary widely between foods and depend on the composition of the food as well as numerous environmental factors. The toxicological significance of lipid oxidation in foods is complicated by interactions of secondary lipid oxidation products with other food components. These interactions could either form complexes that limit the bioavailability of lipid breakdown products or can lead to the formation of toxic products derived from non-lipid sources. A lack of gross pathological consequences has generally been observed in animals fed oxidized fats. On the other hand, secondary products of lipid autoxidation can be absorbed and may cause an increase in oxidative stress and deleterious changes in lipoprotein and platelet metabolism. The presence of reactive lipid oxidation products in foods needs more systematic research in terms of complexities of food component interactions and the metabolic processing of these compounds.     

Formation of highly reactive gamma-ketoaldehydes (neuroketals) as products of the neuroprostane pathway.

Neuroprostanes are prostaglandin-like compounds produced by free radical-induced peroxidation of docosahexaenoic acid, which is highly enriched in the brain. We previously described the formation of highly reactive gamma-ketoaldehydes (isoketals) as products of the isoprostane pathway of free radical-induced peroxidation of arachidonic acid. We therefore explored whether isoketal-like compounds (neuroketals) are also formed via the neuroprostane pathway. Utilizing mass spectrometric analyses, neuroketals were found to be formed in abundance in vitro during oxidation of docosahexaenoic acid and were formed in greater abundance than isoketals during co-oxidation of docosahexaenoic and arachidonic acid. Neuroketals were shown to rapidly adduct to lysine, forming lactam and Schiff base adducts. Neuroketal lysyl-lactam protein adducts were detected in nonoxidized rat brain synaptosomes at a level of 0.09 ng/mg of protein, which increased 19-fold following oxidation in vitro. Neuroketal lysyl-lactam protein adducts were also detected in vivo in normal human brain at a level of 9.9 +/- 3.7 ng/g of brain tissue. These studies identify a new class of highly reactive molecules that may participate in the formation of protein adducts and protein-protein cross-links in neurodegenerative diseases and contribute to the injurious effects of other oxidative pathologies in the brain.