A critical evaluation of the effect of sorbitol on the ferric-xylenol orange hydroperoxide assay.

Measurement of hydroperoxide concentration by the ferric-xylenol orange assay has the advantages of simplicity, convenience, and inertness to oxygen (Gay, C., et al. (1999) Anal. Biochem. 273, 149-155). However, its sensitivity is limited by the molar absorption coefficients, which are less than 50,000 M(-1) cm(-1) for most hydroperoxides. An earlier report showed that this could be significantly enhanced by the inclusion of 100 mM sorbitol in the assay solution, resulting in an increase of the apparent absorption coefficient for H(2)O(2) from 4.46 x 10(4) to 2.24 x 10(5) M(-1) cm(-1) (Wolff, S. P. (1994) Methods Enzymol. 233, 182-189). It was claimed that the technique was also valid for other hydroperoxides, such as butyl and cumyl. In an attempt to extend this modification to a wider range of hydroperoxides, we have confirmed the enhancement of the assay of H2O2 by sorbitol. However, the sensitivity of the measurements of butyl, cumyl, amino acid, protein, and human blood serum hydroperoxides was only approximately doubled by the inclusion of sorbitol. A mechanism explaining the difference in the assay of H2O2 and the organic hydroperoxides is proposed.

Peroxidation of proteins before lipids in U937 cells exposed to peroxyl radicals.

This study provides the first report of the formation of protein hydroperoxides in cells attacked by reactive oxygen species. U937 cells exposed to peroxyl radicals generated by the thermal decomposition of a water-soluble azo compound gradually accumulated hydroperoxide (-OOH) groups. In an incubation for 22 h, 1.2 mM peroxyl radicals was generated and each cell acquired 1.5x10(8) -OOH groups. These groups were located on the cell proteins; no lipid peroxidation was detected. The extent of protein peroxidation was proportional to the rate of generation of the peroxyl radicals. There was no lag period before the onset of peroxidation, indicating that cell antioxidants could not protect the proteins. The half-life of protein hydroperoxides in cell suspensions was approx. 4 h at 37 degrees C. Our results suggest that protein hydroperoxides might have a significant role as intermediates in the development of biological damage initiated by reactive oxygen species.

Peroxidation of proteins and lipids in suspensions of liposomes, in blood serum, and in mouse myeloma cells.

There is growing evidence that proteins are early targets of reactive oxygen species, and that the altered proteins can in turn damage other biomolecules. In this study, we measured the effects of proteins on the oxidation of liposome phospholipid membranes, and the formation of protein hydroperoxides in serum and in cultured cells exposed to radiation-generated hydroxyl free radicals. Lysozyme, which did not affect liposome stability, gave 50% protection when present at 0.3 mg/ml, and virtually completely prevented lipid oxidation at 10 mg/ml. When human blood serum was irradiated, lipids were oxidized only after the destruction of ascorbate. In contrast, peroxidation of proteins proceeded immediately. Protein hydroperoxides were also generated without a lag period in hybrid mouse myeloma cells, while at the same time no lipid peroxides formed. These results are consistent with the theory that, under physiological conditions, lipid membranes are likely to be effectively protected from randomly-generated hydroxyl radicals by proteins, and that protein peroxyl radicals and hydroperoxides may constitute an important hazard to biological systems under oxidative stress.

Determination of iron in solutions with the ferric-xylenol orange complex.

Formation of a colored complex between ferric iron and xylenol orange has been used in a variety of applications, but there is disagreement about the number of complexes that exist, their stoichiometry, pH dependence, solvent effects, and other parameters. In this study we investigated the use of the complex to measure micromolar concentrations of iron in aqueous and alcohol solutions. The results show that the optimum wavelength for the measurement is 560 nm, that only a 1:1 ferric:xylenol orange complex forms, and that its molar absorption coefficient is affected by the solvent, the source of the xylenol orange, and the pH. Under the recommended conditions, formation of the complex is completed in less than 5 min and it is stable in several solvents. Its molar absorption coefficient in 25 mM H(2)SO(4) is 20,100 or 14,500 M(-1) cm(-1), depending on the source of the dye. A recommended protocol for the use of the assay is given.

Hydroperoxide assay with the ferric-xylenol orange complex.

A range of hydroperoxides were reduced by ferrous ions in acid solutions and the amount of ferric product was measured as a xylenol orange complex at 560 nm. Dilute sulfuric acid, 50% acetic acid, and acidified 90% methanol proved to be suitable solvents. The color developed within 15 min and was stable for several hours in most solvents. The apparent molar absorption coefficients (epsilon(app)) of H(2)O(2) and of the t-butyl, cumene, bovine serum albumin, and linoleate hydroperoxides were measured, using known hydroperoxide concentrations determined independently by an iodometric assay. The epsilon(app) values differed significantly and depended on the hydroperoxide, the solvent, and the source of the xylenol orange. The numbers of Fe(3+) ions formed by a range of hydroperoxides in different solvents showed that H(2)O(2) gave 2.5, t-butyl and cumene hydroperoxides 5, and the other hydroperoxides 2 Fe(3+) ions per -OOH group. This general finding allows the determination of approximate hydroperoxide concentrations even in chemically complex systems. Accurate measurements require knowledge of the nature of the hydroperoxide and its epsilon(app) and careful control of the assay conditions. However, the convenience of the assay makes it potentially useful in a variety of applications.

Decomposition of lipid hydroperoxides enhances the uptake of low density lipoprotein by macrophages.

This study examined the roles of low-density lipoprotein (LDL) lipid oxidation and peroxide breakdown in its conversion to a form rapidly taken up by mouse peritoneal macrophages. Oxidation of the LDL without decomposition of the hydroperoxide groups was performed by exposure to gamma radiation in air-saturated solutions. Virtually complete decomposition of the hydroperoxides was achieved by treatment of the irradiated LDL with Cu2+ under strictly anaerobic conditions. No uncontrolled LDL uptake by macrophages occurred when the lipoprotein contained less than 150 hydroperoxide groups per particle. More extensively oxidized LDL was taken up and degraded by mouse macrophages significantly faster than the native lipoprotein. The uptake was greatly enhanced by treatment of the oxidized LDL with Cu2+. A significant proportion of the LDL containing intact or copper-decomposed LDL hydroperoxide groups accumulated within the macrophages without further degradation. Treatment of the radiation-oxidized LDL with Cu2+ was accompanied by aggregation of the particles. Competition studies showed that the oxidized LDL was taken up by macrophages via both the LDL and the scavenger receptors, whereas the copper-treated lipoprotein entered the cells only by the scavenger pathway. Phagocytosis also played an important role in the metabolism of all forms of the extensively modified LDL. Our results suggest that minimally-oxidized LDL is not recognized by the macrophage scavenger receptors unless the lipid hydroperoxide groups are decomposed to products able to derivatize the apo B protein.

Crosslinking of DNA and proteins induced by protein hydroperoxides.

Exposure of DNA to several proteins peroxidized by radiation-generated hydroxyl free radicals resulted in formation of crosslinks between the macromolecules, detected by retardation and broadening of DNA bands in agarose gels. This technique proved suitable for the study of crosslinking of DNA with peroxidized BSA, insulin, apotransferrin and alpha casein, but not with several other proteins, including histones. The crosslinking depended on the presence of intact hydroperoxide groups on the protein, on their number, and on the duration of the interaction with DNA. All DNA samples tested, pBR322, pGEM, lambda/HindIII and pUC18, formed crosslinks with the peroxidized BSA. Sodium chloride and formate prevented the crosslinking if present during incubation of the peroxidized protein and DNA, but had no effect once the crosslinks had formed. The gel shift of the crosslinked DNA was reversed by proteolysis, indicating that the DNA mobility change was due to attachment of protein and that the crosslinking did not induce DNA strand breaks. The metal chelators Desferal and neocuproine reduced the extent of the crosslinking, but did not prevent it. Scavengers of free radicals did not inhibit the crosslink formation. The DNA-protein complex was not disrupted by vigorous agitation, by filtration or by non-ionic detergents. These observations show that the crosslinking of DNA with proteins mediated by protein hydroperoxides is spontaneous and probably covalent, and that it may be assisted by transition metals. It is suggested that formation of such crosslinks in living organisms could account for some of the well-documented forms of biological damage induced by reactive oxygen species-induced oxidative stress.

Antiviral activity of 1,7-disulphoanthraquinone.

The antiviral activity of 1,7-disulphoanthraquinone (DSA) against hepatitis B viruses was investigated by measuring the titer of HBV surface antigen in the treated serum obtained from the blood of patients with acute infection. The presence of HBsAg was tested by the Vitek Immuno Diagnostic Assay System (VIDAS). The concentration of DSA in the samples was equal to 0.01, 0.1 and 1%, respectively. The results presented clearly showed an extensive disintegration of the virus envelope at elevated temperatures, which resulted in a substantial decrease in the concentration of HBsAg in the serum containing DSA. The concentration of HBsAg decreased also upon UV irradiation of the serum containing DSA in a photochemical reactor for 5 to 15 min, but the effect of degradation was not complete.

Protein hydroperoxides as new reactive oxygen species.

There is convincing evidence for the involvement of Reactive Oxygen Species (ROS) in the initiation and development of various forms of damage in living organisms. Attempts to prevent or limit such damage have been largely unsuccessful, principally because most of the pathways linking the formation of ROS with the end-point pathology are unknown. Evidence summarized in this review suggests that proteins are the most likely initial targets of ROS in cells and that protein hydroperoxides are major products of this interaction. Recent research has shown that the protein hydroperoxides can in turn generate new free radicals, inactivate enzymes, destroy antioxidants, and crosslink with DNA. This suggests that protein hydroperoxides may constitute an important intermediate stage in the development of ROS-induced biological damage, and that they should therefore be regarded as a new form of reactive oxygen species.

The limitations of an iodometric aerobic assay for peroxides.

A technique recommended for the assay of lipid and other organic peroxides based on the use of a commercial color reagent (El-Saadani et al., J. Lipid Res. 30, 627-630, 1989) has the advantage over other iodometric methods of being insensitive to oxygen. Although tested so far with a limited range of peroxides, this aerobic method has found popular use with complex biological systems, such as plasma. We have examined the ability of this assay to provide accurate estimates of peroxides in H2O2, tert-butanol, and cumene hydroperoxides, and in oxidized linoleate, low-density lipoprotein, and human blood plasma. The results were compared with values obtained with an anaerobic iodometric peroxide method taken as the standard peroxide assay. We found that the published protocol gave correct peroxide values for H2O2 solutions. Correct values could also be obtained for oxidized low-density lipoprotein, provided that the incubation period was extended from 30 to 60 min. All the other peroxides tested gave much lower values than those of the standard iodometric method. Incubation at 50 degrees C to increase the velocity of the reaction for some of the slowly reacting peroxides did not improve the accuracy of the aerobic method. We recommend that the color reagent should be used as originally specified only for the assay of H2O2, or for oxidized lipoprotein with the incubation extended to 60 min.

Effect of amino acid peroxides on the erythrocyte.

The effect of amino acid peroxides, relatively stable products of irradiation of amino acid solutions, on erythrocyte components was studied. Interaction of proline, lysine, valine, and leucine peroxides (100-300 mu M) with erythrocyte membranes brought about a decrease of membrane protein -SH group content and of activities of (Na+, K+)-ATPase and Ca2+ -ATPase, and induced aggregation of membrane proteins, due mainly to the formation of interpeptide disulfides. Interaction of amino acid peroxides with hemoglobin brought about hemoglobin oxidation to methemoglobin. The effects of amino acid peroxides are similar to those of t-butyl hydroperoxide. These results indicate that peroxides of amino acid and proteins, which can also be formed under physiological conditions, may be mediators of the cellular action of reactive oxygen species.

Biological fate of amino acid, peptide and protein hydroperoxides.

In the course of searching for a suitable marker for studying protein oxidation, we have successfully elucidated the structures of three valine hydroperoxides, i.e. beta-hydroperoxyvaline, (2S,3S)-gamma-hydroperoxyvaline and (2S,3R)-gamma-hydroperoxyvaline, which are novel products of protein oxidation. The corresponding valine hydroxides were obtained by sodium borohydride reduction [Fu, Hick, Sheil and Dean (1995) Free Rad. Biol. Med. 19, 281-292]. We hypothesized that valine hydroxides might be the major biological degradation products of valine hydroperoxides and, as such, could be useful markers for the study of protein oxidation in vivo. The aim of this study was to investigate the fate of valine hydroperoxide in selected biological systems by the use of chemiluminescence detection of hydroperoxides and HPLC analysis of O-phthaldialdehyde derivatives of amino acid residues. The degradation of hydroperoxides present on gamma-radiolysed solutions of valine, Pro-Val-Gly, or BSA occurred in the presence of: (1) transition metals (Fe2+, Fe3+, or Cu2+), (2) the detoxifying enzyme GSH peroxidase, (3) human plasma, and (4) J774 mouse monocyte macrophage cells. The major degradation product of valine hydroperoxide recovered in each case was found to be a valine hydroxide. These results suggest that valine hydroxide (derived from the hydroperoxide) may well be a useful in vivo marker for studying protein damage under oxidative stress.

Stimulation of erythrocyte membrane Mg(2+)-ATPase activity by dinitrophenol and other membrane-disturbing agents.

Erythrocyte membrane Mg(2+)-ATPase activity was stimulated by echinocytogenic agents (2,4-dinitrophenol and salicylate), a stomatocytogenic agent Triton X-100 and other membrane-disturbing agents including hydrophobic organic anions, alcohols and detergents. Various possible mechanisms of the stimulation are possible but apparently most probable one consists in induction of membrane phospholipid scrambling by the compounds studied (as demonstrated for DNP) and of aminophospholipid translocase (flippase) activity.

Continuous measurement of oxygen consumption by linoleic acid membranes exposed to free radicals generated by gamma-radiation.

Vesicles enclosed by membranes prepared from linoleic acid were exposed in the chamber of an oxygen electrode to free radicals generated by 60Co gamma-rays. Oxidation was observed by oxygen consumption, conjugated diene formation, and tri-iodide assay for hydroperoxides. There was a dose-dependent lag period before the onset of rapid peroxidation. The radiation chemical yields (G-values) ranged from 4.45 to 19.37 mu-mol J-1 for maximum rates of oxygen consumption and from 2.18 to 16.37 mumol J-1 for maximum rates of hydroperoxide production when the radiation dose-rate was varied between 5.39 and 0.14 Gy min-1. The magnitudes of these G-values and the linear relationship between yield of hydroperoxide and (dose-rate)1/2 were indicative of a chain mechanism for peroxidation operating in membranes. The lack of congruence between the amount of oxygen consumed and hydroperoxide formed suggested that the oxygen consumed in membrane oxidation led to the formation of oxidized derivatives of linoleic acid additional to the hydroperoxides.

Radical-induced chain oxidation of proteins and its inhibition by chain-breaking antioxidants.

Exposure of proteins to oxygen-centred radicals results in dramatic changes in their structure, stability and function; properties that have been studied in many laboratories from a qualitative viewpoint. To allow a quantitative evaluation, we subjected aerated solutions of BSA to hydroxyl and superoxide anion radicals generated radiolytically under conditions where all radicals formed reacted with the protein (as judged by maximum damage to BSA). We observed that for each radical generated approx. 15 amino acids were consumed initially. Similar results were found with lysozyme and melittin. Such a massive consumption of BSA's amino acids was not observed when irradiations were carried out under anaerobic conditions. When bilirubin or Trolox (a water-soluble analogue of vitamin E) was added at a 2-fold molar excess over BSA, initial consumption of all measured amino acids, except tryptophan, decreased 4-fold. The total mass of amino acids initially protected (from consumption) exceeded the mass of antioxidants consumed by more than a factor of 20. Such protection of amino acids was not observed when the antioxidant-inactive acetyl Trolox was used. These results suggest that radical-mediated oxidation of proteins can proceed via a previously unrecognized chain reaction that may be inhibited by chain-breaking antioxidants.