Melinda R. Vinqvist

MEMBRANE PEROXIDATION : INHIBITING EFFECTS OF WATER-SOLUBLE ANTIOXIDANTS ON PHOSPHOLIPIDS OF DIFFERENT CHARGE TYPES

Quantitative kinetic methods of autoxidation are used to determine the antioxidant activities of two water-soluble antioxidatants of the chromanol type, 6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid (Trolox) and 6-hydroxy-2,5,7,8- tetramethyl-2-N,N,N-trimethylethanaminium methylbenzene-sulfonate (MDL 73404), during free radical peroxidation of phospholipid membranes of different charge types. The stoichiometric factor (n) for peroxyl radical trapping for both Trolox and MDL 73404 was found to be 2. Trolox was found to partition partially, approximately 20%, into the lipid phase of liposomes. The antioxidant activity of Trolox during peroxidation of membranes determined by measurements of the absolute rate constant for inhibition of oxygen uptake, kinh, was found to vary with the membrane surface charge that is controlled by variation in pH. When peroxidation is initiated in the lipid phase by azo-bis-2,4- dimethylvaleronitrile (ADVN), using a typical zwitterionic liposome, dilinoleoylphosphatidyl choline (DLPC), the kinh was found to be 2.98 X 10(3) M-1s-1. The kinh of Trolox increased approximately 2-fold for membranes that have a positive surface, including DLPC at pH 4, DLPC containing stearylamine at pH 7, and for a membrane of dimyristoylphosphatidyl acid containing linoleic acid (DMPA/LA). Conversely, Trolox does not inhibit peroxidation of negatively charged dilinoleoylphosphatidyl glycerol (DLPG) at pH 7-11. Studies made of the positively charged MDL 73404 show that its antioxidant activity using DLPC and DLPG is pH dependent. Trolox inhibits the peroxidations of DLPC initiated in the aqueous phase by azo-bis-(2-amidinopropane-HCl)(ABAP) at pH 4 or 7. However, Trolox does not inhibit the peroxidation of DLPG at pH 7. The different antioxidant activities of Trolox and MDL 73404 are rationalized in terms of a peroxyl-radical diffusion model and specific charge interactions between antioxidants and membrane surface.

Cholesterol : Free radical peroxidation and transfer into phospholipid membranes

Cholesterol, when sequestered in saturated liposomes of dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC), undergoes peroxidation thermally initiated either by a lipid-soluble or a water-soluble azo initiator and in both cases the reaction is inhibited effectively by the water-soluble antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (Trolox). Quantitative kinetic methods of autoxidation show that the oxidizability, kp/(2kt)1/2 (where kp and 2kt are the rate constants of radical chain propagation and termination, respectively) of cholesterol in DMPC or DPPC multilamellar liposomes, where kp/(2kt)1/2 is 3.0.10(-3) to 4.3.10(-3) M-1/2 s-1/2 at 37-45 degrees C, is similar to that measured in homogeneous solution in chlorobenzene, where kp/(2kt)1/2 is 3.32.10(-3). However, its oxidizability in smaller unilamellar vesicles of DMPC or DPPC increases by at least 3-times that measured in multilamellar systems. Autoxidation/antioxidant methods show that cholesterol partitions directly from the solid state into DMPC or DPPC liposomes by shaking and this is confirmed by 31P and 2H quadrupole NMR spectra of deuterated cholesterol when membrane bound. Analytical studies indicate that up to 21 mol% cholesterol will partition into the membranes by shaking.

The efficiency of antioxidants delivered by liposomal transfer

Phenolic antioxidants of the hydroxychroman class, alpha-tocopherol (alpha-TOC) and 2,2,5,6,7-pentamethyl-6-hydroxychroman (PMHC), and the hindered phenols 2,3-dihydro-5-hydroxy-2,2,4-trimethylnaphtho[1,2-b]furan (NFUR), 2,6-di-tert-butyl-4-methoxyphenol (DBHA), and 2,6-di-tert-butyl-4-methyl phenol (BHT), were delivered into oxidizable (ACCEPTOR) liposomes of dilinoleoylphosphatidylcholine (DLPC) or 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) from saturated DONOR liposomes of dimyristoylphosphatidylcholine (DMPC) by liposomal transfer. The antioxidant activities, k(inh), by the inhibited oxygen uptake method were compared with the k(inh)s determined when the antioxidants were introduced into the liposomes by coevaporation from organic solvents. The peroxidations were initiated using either thermal initiators, water-soluble azo-bis-amidinopropane hydrochloride (ABAP), lipid-soluble azo-bis-2,4-dimethylvaleronitrile (ADVN) and di-tert-butylhyponitrite (DBHN), or the photoinitiator benzophenone. The antioxidants PMHC, NFUR, DBHA, and BHT transferred rapidly between liposomes, but several hours of incubation were needed to transfer alpha-TOC. The average k(inh)s in liposomes, in the relative order NFUR approximately DBHA > PMHC > BHT approximately alpha-TOC, were markedly lower than known values in organic solvent. k(inh) values in liposomes appear to be controlled by effects of hydrogen bonding with water and by restricted diffusion of antioxidants, especially in the case of alpha-TOC. Product studies of the hydroperoxides formed during inhibited oxygen consumption were carried out. The cis,trans/trans,trans (c,t/t,t) product ratios of the 9- and 13-hydroperoxides formed from PLPC during inhibited peroxidation by PMHC were similar for both the coevaporated and liposomal transfer procedures. The c,t/t,t ratio for the same concentration of alpha-TOC, 1.52, compares to a value of 1.69 for PMHC at the start of the inhibition period. The higher c,t/t,t ratio observed for NFUR in DLPC, which varied between values of 7.0 at the start of the inhibition to about 1.8 after the break in the induction period, is a reflection of the increased hydrogen atom donating ability of the antioxidant plus the increased concentration of oxidizable lipid provided by DLPC.

Photooxidations Initiated or Sensitized by Biological Molecules: Singlet Oxygen Versus Radical Peroxidation in Micelles and Human Blood Plasma ¶

Biomolecules common in blood plasma, including 2‐methyl‐1,4‐naphthoquinone (vitamin K‐0, 2), 2,3‐dimethoxy‐5‐methyl‐1,4‐benzoquinone (ubiquinone‐0, 3), bilirubin, 4, and urocanic acid, 5, were used as photoactivators for the photooxidation of methyl linoleate (ML) in 0.50 M sodium dodecyl sulfate micelles to mimic a bioenvironment. UV irradiation of 2 in this system initiated H‐atom abstraction from ML (Type‐I mechanism). The evidence includes kinetics of oxygen uptake, inhibition of oxidation by an antioxidant ((R)‐(+)‐6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid [Trolox], 7) and the analysis of four geometric hydroperoxides formed (cis, trans to trans, trans ratio of 0.5). In contrast, irradiation with a singlet‐oxygen sensitizer, 3,5‐di‐t‐butyl‐1,2‐benzoquinone, 1, formed six isomers by a Type‐II mechanism, yielding a cis, trans to trans, trans isomer ratio of 6. Peroxidation activated by 3 or 4 with visible light occurred by a singlet‐oxygen pathway (Type‐II mechanism), as shown by kinetics of oxygen uptake and the effect of quenchers. In contrast, peroxidation in the presence of 5 in this system initiated H‐atom abstraction from ML as shown by oxygen uptake and inhibition by Trolox. A comparison of thermal free‐radical peroxidation with direct photooxidation of human blood plasma samples showed important differences. Blood plasma resisted thermal peroxidation because of natural antioxidants or on the addition of Trolox. In contrast, direct photooxidation involved singlet oxygen, according to the effect of quenchers and the lack of inhibition by antioxidants.