The effects of alpha-tocopherol on site-specific lipid peroxidation induced by iron in charged micelles

alpha-Tocopherol inhibited H2O2-Fe2+-induced lipid peroxidation of linoleic acid (LA) by scavenging OH radicals in tetradecyltrimethylammonium bromide (TTAB) micelles. The inhibiting ability of alpha-tocopherol was much greater than that of OH-radical scavengers mannitol and t-butanol. In contrast, alpha-tocopherol enhanced linoleic acid hydroperoxide (LOOH)-Fe2+-induced lipid peroxidation through regeneration of Fe2+ in sodium dodecyl sulfate (SDS) micelles containing LA. alpha-Tocopherol was oxidized by Fenton's reagent (FeSO4 + H2O2) at a higher rate in SDS micelles than in TTAB micelles. The likely oxidants were OH radicals in the former and Fe3+ in the latter. Both reagents formed in the Fenton reaction. Ferrous ion catalyzed in a dose-dependent manner the decomposition of LOOH and conjugated diene compounds in SDS but not in TTAB micelles. alpha-Tocopherol and Fe3+ individually had no effect on the decomposition of LOOH, but together were quite effective. The rate of the decomposition was a function of the concentration of alpha-tocopherol. The mechanism of "site-specific" antioxidant action of alpha-tocopherol in charged micelles is discussed.     

The effect of concentration on the antioxidant effectiveness of alpha-tocopherol in lipid peroxidation induced by superoxide free radicals

Egg yolk phosphatidylcholine liposomes were rapidly oxidized in the presence of chelated iron and a superoxide-generating system. alpha-Tocopherol incorporated in the bilayer was oxidized at the same time. No lipid or alpha-tocopherol oxidation occurred in liposomes composed of dimyristoyl phosphatidylcholine. The antioxidant did not inhibit lipid peroxidation until its concentration reached a critical level, which depended on the effectiveness of the oxidative stress. Beyond this level, peroxidation was inhibited completely and, simultaneously, the rate of oxidation of tocopherol was lowered. The results suggest that the antioxidant efficiency of alpha-tocopherol depends on its ability to react mainly with the chain-initiating or chain-propagating lipid radicals. This, in turn, is closely tied to the tocopherol content of the membrane. Ascorbate inhibited the consumption of alpha-tocopherol, possibly by regenerating its reduced form.     

Possible influence of lysophospholipase on the production of 1-acyl-2-acetylglycerophosphocholine in macrophages.

The rate of production of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylPAF) was measured in macrophages following the incorporation of [3H]acetate. Upon activation by A23187, guinea pig alveolar macrophages incorporated [3H]acetate into PAF, but a little radioactivity was found in acylPAF. However, labeling of acylPAF and PAF with [3H]acetate was greatly enhanced in A23187-stimulated alveolar macrophages that had been pretreated with phenylmethanesulphonyl fluoride (PMSF). [3H]PAF was predominantly converted to 1-[3H]alkyl-2-acyl glycerophosphocholine, but [14C]acylPAF rapidly hydrolyzed to 14C-labeled free fatty acid by the incubation with lysates prepared from macrophages. The deacetylation of [14C]acylPAF and [3H]PAF by acetylhydrolase and also the hydrolysis of [14C]lysoPC by lysophospholipase were strongly inhibited in macrophages that had been pretreated with PMSF, while PMSF failed to inhibit the activities of acetyltransferase and acyltransferase. The relative proportions of PAF and acylPAF were quite different in different types of cells. In contrast to alveolar macrophages, peritoneal macrophages, neutrophils and spleen cells from guinea pigs incorporated 2-4 times more [3H]acetate into acylPAF than into PAF. The presence of high levels of acylPAF in peritoneal macrophages was confirmed by GLC-MS analysis. The activities of lysophospholipase, acetylhydrolase and acetyltransferase were measured in alveolar and peritoneal macrophages to determine whether the preferential formation of acylPAF as compared to PAF in peritoneal macrophages was due to differences in these activities between alveolar and peritoneal macrophages. The activity of acetylhydrolase of peritoneal macrophages was almost the same as that in alveolar macrophages. The activity of acetyltransferase in peritoneal macrophages was about half of that in alveolar macrophages. However, the activity of lysophospholipase in peritoneal macrophages was one-sixth of that in alveolar macrophages. These results suggest that lysophospholipase is one of the primary factors involved in the control of the production of acylPAF in activated cells, and that it acts by modulating the availability of lysoPC for the synthesis of acylPAF. Furthermore, high levels of activity of lysophospholipase allow the preferential formation of PAF, via the rapid hydrolysis of lysoPC which would act as a competitive inhibitor of the incorporation of acetate into lysoPAF.     

Translocation of exogenous platelet-activating factor and its lyso-compound through plasma membranes is a rate-limiting step for their metabolic conversions into alkylacylglycerophosphocholines in rabbit platelets and guinea-pig leukocytes

Platelets and leukocytes are known to degrade platelet-activating factor (PAF), a potential mediator of inflammation, to its lyso-derivative (lyso-PAF) and then convert this to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines. However, little is known about the mechanism of internalization of PAF and lyso-PAF, which is a prerequisite for their metabolism within the cells. In this work, the internalization of PAF and lyso-PAF by rabbit platelet and guinea-pig leukocyte plasma-membranes were examined by the washing method with bovine serum albumin. The rates of translocation of PAF and lyso-PAF across guinea-pig plasma membranes were significantly higher than those across rabbit platelets. In these cells, the translocation of PAF was found to be accelerated indirectly by activation of PAF receptors by a small portion of added PAF. Results suggest that a temperature-dependent diffusion process is involved in the internalization of these phospholipids. In both rabbit platelets and guinea-pig leukocytes, the translocation of PAF and lyso-PAF through the plasma membranes was shown to be rate-limiting for the metabolic conversion of these compounds to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine.     

Antioxidative effect of α-tocopherol incorporation into lecithin liposomes on ascorbic acid-Fe2+-induced lipid peroxidation

The antioxidative effect of α-tocopherol incorporated into lecithin liposomes was studied. Lipid peroxidation of liposome membranes, assayed as malondialdehyde production, was catalyzed by ascorbic acid and Fe²⁺. The peroxidation reaction, which did not involve the formation of singlet oxygen, superoxide, hydrogen peroxide, or a hydroxyl radical, was inhibited by α-tocopherol and a model compound of α-tocopherol, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (TMC), but not by phytol, α-tocopherylquinone, or α-tocopheryl acetate. One mole of α-tocopherol completely prevented peroxidation of about 100 moles of polyunsaturated fatty acid. Decrease in membrane fluidity by lipid peroxidation, estimated as increase of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in the membrane, was also inhibited by α-tocopherol and TMC, reflecting their antioxidant functions. Cholesterol did not act as an antioxidant, even when incorporated in large amount into the liposome membranes, but it increased the antioxidative efficiency of α-tocopherol. When a mixture of liposomes with and without α-tocopherol was incubated with Fe²⁺ and ascorbic acid, α-tocopherol did not protect the liposomes not containing α-tocopherol from peroxidation. However, preincubation of the mixture, or addition of Triton X-100 allowed the α-tocopherol to prevent peroxidation of the liposomes not containing α-tocopherol. In contrast, in similar experiments, liposomes containing TMC prevented peroxidation of those without TMC without preincubation. Tocopherol in an amount so small as to exhibit only a slight antioxidative effect was oxidized when incorporated in egg lecithin liposomes, but it mostly remained unoxidized when incorporated in dipalmitoyllecithin liposomes, indicating that oxygen activated by ascorbic acid-Fe²⁺ does not oxidize α-tocopherol directly. Thus, decomposition of α-tocopherol may be caused by its interaction with peroxy and/or alkoxyl radicals generated in the process of lipid peroxidation catalyzed by Fe²⁺ and ascorbic acid.     

Transbilayer movement and metabolic fate of ether-linked phosphatidic acid (1-O-Octadecyl-2-acetyl-sn-glycerol 3-phosphate) in guinea pig peritoneal polymorphonuclear leukocytes.

1-O-Octadecyl-2-acetyl-sn-glycerol 3-phosphate (octadecylacetyl-GP) and its deacetylation product were used as a model of phosphatidic acid and its lyso derivatives, respectively. The binding, transbilayer movement, and intermembranous transport, which should be related to its metabolism in guinea pig peritoneal polymorphonuclear leukocytes, were studied. The albumin extraction procedure (Tokumura, A., Tsutsumi, T., Yoshida, J., and Tsukatani, H. (1990) Biochim. Biophys. Acta 1044, 91-100) was used for studying the transbilayer movement of [3H]octadecylacetyl-GP. The binding, translocation, and metabolism of octadecylacetylglycerol, a dephosphorylated product of octadecylacetyl-GP, in polymorphonuclear leukocytes were also investigated for comparison. The translocation of octadecylacetyl-GP was dependent on temperature, but not on its concentration (in the range of 1-100 nM). The rate of translocation of octadecylacetyl-GP was much slower than that of octadecylacetylglycerol. Treatment of polymorphonuclear leukocytes with N-ethylmaleimide did not affect the translocation of octadecylacetyl-GP. These results suggest that the transbilayer movement of octadecylacetyl-GP is driven by a diffusion process, not by a carrier protein. From these findings, the process of translocation of octadecylacetyl-GP is concluded to be a rate-limiting step in its metabolic conversion to triglyceride, phosphatidylethanolamine, and phosphatidylcholine.     

Metabolism of benzoquinone by yeast cells and oxidative characteristics of corresponding hydroquinone: application to highly sensitive measurement of yeast cell density by using benzoquinone and a chemiluminescent probe

The metabolic efficiency of seven derivatives of 1,4-benzoquinone (BQ) by yeast cells and the oxidative characteristics of the corresponding hydroquinones (HQs) were studied by electrochemical, spectrophotometric and chemiluminescent methods. The spectrophotometric method was based on the reduction of a tetrazolium salt to formazan dye during the autoxidation of HQs generated by yeast cells under alkaline conditions. The amounts of HQs detected directly by the electrochemical method did not agree with those calculated from the formazan dye obtained by the spectrophotometric method. A tetrazolium salt was reduced to a formazan dye by both the superoxide anion radical (O2-*) generated during the autoxidation of 2,3,5,6-tetramethyl-1,4-HQ and by HQ itself. Little formazan dye was formed, and hydrogen peroxide (H2O2) was then finally produced during the autoxidation of 1,4-HQ or 2-methyl-1,4-HQ. Formazan dye and H2O2 were generated at a certain ratio during the autoxidation of derivatives of dimethyl-1,4-HQ or 2,3,5-trimethyl-1,4-HQ. The analytical method based on chemiluminescence with lucigenin and 2,3,5,6-tetramethyl-1,4-BQ was applied to highly sensitive measurement of the yeast cell density. A linear relationship between the chemiluminescence intensity and viable cell density was obtained in the range of 1.2 x 10(3) - 4.8 x 10(4) cells/ml. The detection limit was 4.8 x 10(2) cells/ml.     

Potential changes with gamma-band oscillation at the frontal scalp elicited by intravenous olfactory stimulation in humans

Intravenous olfaction is a unique stimulation method often used in Japan to diagnose olfactory disturbances. Odorant is injected into a vein and transported by blood flow and respiration to the upper air tract. The intravenous olfaction might allow the potential at the frontal scalp to be recorded without contamination from electromyograms, such as those caused by sniffing. We injected Alinamin (thiamine propyldisulphide) into healthy subjects according to a standard protocol for clinical intravenous olfaction testing and we simultaneously recorded potential changes at the frontal scalp. When Alinamin was injected into the right median cubital vein over a 20 s period, the potential changes with gamma-band oscillations were detected 17.6 +/- 6.7 s (mean +/- SD) after the start of the injection. The main frequency component of this gamma-band oscillation is 30-160 Hz. The gamma-band oscillation elicited by intravenous olfactory stimulation (VOP) was similar to the induced wave of the olfactory bulb. Mapping the VOPs on the frontal scalp of a subject with less developed frontal sinuses and the relation between the thickness of the frontal sinuses and VOP amplitude suggest an intracranial source, possibly the olfactory bulb. The gamma-band potential at the frontal scalp is a useful measure of central disturbance.