Janusz M. Gebicki

The role of lipid peroxidation and antioxidants in oxidative modification of LDL.

The purpose of this study is to provide a comprehensive survey on the compositional properties of LDL (e.g., lipid classes, fatty acids, antioxidants) relevant for its susceptibility to oxidation, on the mechanism and kinetics of LDL oxidation, and on the chemical and physico-chemical properties of LDL oxidized by exposure to copper ions. Studies on the occurrence of oxidized LDL in plasma, arteries, and plaques of humans and experimental animals are discussed with particular focus on the use of poly- and monoclonal antibodies for immunochemical demonstration of apolipoprotein B modifications characteristic for lipid peroxidation. Apart from uptake of oxidized LDL by macrophages, studies describing biological effects of heavily or minimally oxidized LDL are only briefly addressed, since several reviews dealing with this subject were recently published. This article is concluded with a section on the role of natural and synthetic antioxidants in protecting LDL against oxidation, as well as some previously unpublished material from our laboratories.

A spectrophotometric method for the determination of lipid hydroperoxides

A modification of the iodine technique for peroxide determination is described, in which the reaction is carried out in deaerated methanol:acetic acid solution in a spectrophotometer cuvette. Measurements of the triiodide ions released are carried out either at 290 or 360 nm. In this solvent, the molar extinction coefficients are e 290 = 4.41 × 10 4 and e 360 = 2.80 × 10 4 liters mol −1 cm −1 . Calibration with standard peroxide solutions showed that 1 mol of I 3 − is produced for every peroxide group added. In its present form, the method can be used to measure 1 nEq of lipid hydroperoxide.

Oxidation of α-tocopherol in micelles and liposomes by the hydroxyl, perhydroxyl, and superoxide free radicals☆

Rates of oxidation of alpha-tocopherol by the hydroxyl- and superoxide free radicals were measured. The radicals were produced in known yields by radiolysis of aqueous solutions with gamma rays. Two main systems were used to dissolve the tocopherol; micelles, made up from charged and uncharged amphiphiles, and membranes made from dimyristyl phosphatidylcholine which could be charged by addition of stearyl amine or dicetyl phosphate. The HO. radicals were efficient oxidants of alpha-tocopherol in all systems, with up to 83% of radicals generated in micelle and 32% in membrane suspensions initiating the oxidation. The HO.(2) radical was an even more effective oxidant, but when most of it was in the O(2) form at neutral or alkaline pH, the oxidation rates became low. Tocopherol held in positively charged micelles or membranes was oxidized at a higher rate by the O(2) than in uncharged or negative particles. Possible biological significance of these results is discussed.

Vitamin E content and low density lipoprotein oxidizability induced by free radicals.

Human LDL, HDL and lipoprotein deficient plasma isolated from 15 normal subjects was exposed to oxygen free radicals generated by gamma rays and the formation of peroxides and changes in levels of LDL alpha-tocopherol were measured. LDL exhibited an initial resistance against oxidation stress when compared to HDL. The results obtained for different individuals showed that there was no correlation between the initial levels of vitamin E in LDL or plasma and the amount of peroxide formed after exposure of the LDL to a standard quantity of oxygen radicals. Kinetic experiments with original LDL and LDL containing incorporated alpha-tocopherol demonstrated that the vitamin performed its antioxidant role by conferring some early protection to the lipids, being consumed in the process, but it was clear that additional factors are also instrumental in determining the total antioxidant potential of the human LDL.

Measurement of protein and lipid hydroperoxides in biological systems by the ferric-xylenol orange method

Methods were developed for the separation and measurement of lipid and protein hydroperoxides, which can be used for biological materials. Lipids were extracted with methanol:chloroform and their hydroperoxides measured in solutions of methanol and chloroform containing 110mM perchloric acid, xylenol orange, and ferrous iron. Proteins were isolated by precipitation with 0.2M perchloric acid. The precipitates were redissolved in 6M guanidine hydrochloride and washed with chloroform, and the hydroperoxides were measured in the presence of perchloric acid, xylenol orange, and ferrous iron. Optimum conditions for hydroperoxide measurements were established and the assays were applied to oxidized human blood serum and to cultured cells.

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.

Rate constants for reaction of hydroxyl radicals with Tris, Tricine and Hepes buffers

Rate constants for the reactions of three commonly used organic buffers and hydroxyl radicals were measured using steady‐state competition kinetics with thymine. For Hepes (4‐(2‐hydroxyethyl)‐1‐piperazine‐ethanesulphonic acid), Tricine (N‐[2‐hydroxy‐1,1‐bis(hydroxymethyl)ethyl]glycine and Tris (2‐amino‐2‐hydroxymethylpropane‐1,3‐diol) the rate constants were 5.1×109, 1.6×109 and 1.1 × 109 1·mol−1·s−1, respectively.

The effect of pH on the conversion of superoxide to hydroxyl free radicals

The conversion of superoxide (O-.2) to the hydroxyl (HO.) free radical by superoxide-driven Fenton reactions was measured by the formation of hydroxylated derivatives from benzoate. Among a range of catalysts required for the conversion, the Fe3+EDTA complex was the most effective. The effect of superoxide dismutase and catalase indicated that O-.2 and H2O2 were essential reactants, while the formation of authentic HO. was confirmed by the inhibiting capacities of formate, t-butanol, and mannitol. The conversion of O-.2 to HO. was tested over a broad pH range, and was found to be highest at pH 4.8 whether Fe3+EDTA or free Fe3+ were used as the catalysts. When Fe3+EDTA was used at the optimum pH, every HO. produced required 3.7 O-.2 radicals, close to the theoretical limit of one HO. from every three O-.2 radicals generated.

[29] Iodometric determination of hydroperoxides in lipids and proteins

Publisher Summary This chapter discusses the iodometric determination of hydroperoxides in lipids and proteins. During the autoxidation of lipids, hydroperoxides are formed as major early reaction products. Proteins and amino acids are also sites of hydroperoxide formation during oxidation. Interest in the formation, properties, and metabolism of hydroperoxides in biological systems has led to the development of a number of methods for their determination. In appropriate circumstances—that is, where the decay of hydroperoxides is not significant—their measurement can also be a useful index of early oxidative damage. The outstanding advantage of the iodometric hydroperoxide assay over all other methods is the ability of hydroperoxides in a wide range of molecules to react with iodide quantitatively and with stoichiometry. Other assays may react quantitatively only with specific classes of hydroperoxides, and/or the stoichiometry of the reactions is variable or unknown. The iodometric assay is particularly useful in complex biological systems where hydroperoxides may be generated on a diverse array of molecules.

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.