Presence Of Alpha-tocopherol Research Articles

Inverse dose-rate effects at the level of proteins observed in the presence of lipids

Purpose : Radical-chain mechanisms such as lipid peroxidation are known to show an inverse dose-rate effect, i.e. the radiation effect increases with decreasing dose rate at identical doses applied. The present study was intended to investigate whether an inverse dose-rate effect can be transferred from the level of lipids to the level of proteins. Method : Functional inactivation or structural modification by 80kV X-rays of two classes of proteins was investigated: membrane proteins with a natural environment of lipids like the Ca-ATPase of the sarcoplasmic reticulum, succinate dehydrogenase and F 0 F 1 -ATPase from the inner mitochondrial membrane. The second class comprises the watersoluble proteins cytosolic creatine kinase (MM-CK) and bovine serum albumin (BSA). Their modification by free radicals of water radiolysis was investigated in the absence and presence of lipid vesicles. Results : For all proteins investigated, an inverse dose-rate effect was observed in the presence of lipids. This also holds for the watersoluble proteins MM-CK and BSA. In the latter two cases, the dose-rate effect disappeared either in the absence of (unsaturated) lipids or in the presence of alpha-tocopherol. Conclusion : The largely identical results obtained for a variety of different proteins indicate that inverse dose-rate effects are a normal consequence of radiation induced protein damage in the presence of lipids. In view of the high amount of cellular lipids, this should also hold for the situation in vivo, although due to the comparatively high concentration of intracellular antioxidants the dose-rate dependence might be strongly reduced or even virtually abolished.

Macrophages Can Decrease the Level of Cholesteryl Ester Hydroperoxides in Low Density Lipoprotein

Murine and human macrophages rapidly decreased the level of cholesteryl ester hydroperoxides in low density lipoprotein (LDL) when cultured in media non-permissive for LDL oxidation. This process was proportional to cell number but could not be attributed to the net lipoprotein uptake. Macrophage-mediated loss of lipid hydroperoxides in LDL appears to be metal ion-independent. Degradation of cholesteryl linoleate hydroperoxides was accompanied by accumulation of the corresponding hydroxide as the major product and cholesteryl keto-octadecadienoate as a minor product, although taken together these products could not completely account for the hydroperoxide consumption. Cell-conditioned medium possessed a similar capacity to remove lipid hydroperoxides as seen with cellular monolayers, suggesting that the activity is not an integral component of the cell but is secreted from it. The activity of cell-conditioned medium to lower the level of LDL lipid hydroperoxides is associated with its high molecular weight fraction and is modulated by the availability of free thiol groups. Cell-mediated loss of LDL cholesteryl ester hydroperoxides is facilitated by the presence of alpha-tocopherol in the lipoprotein. Together with our earlier reports on the ability of macrophages to remove peroxides rapidly from oxidized amino acids, peptides, and proteins as well as to clear selectively cholesterol 7-beta-hydroperoxide, results presented in this paper provide evidence of a potential protective activity of the cell against further LDL oxidation by removing reactive peroxide groups in the lipoprotein.

Recent topics in anti‐oxidative factors

Lipids and anti-oxidative factors regulate immune functions such as immunoglobulin production and chemical mediator release. For example, polyunsaturated fatty acids (PUFA) enhance IgE production of isolated rat lymphocytes, but not in the presence of alpha-tocopherol. In addition, anti-oxidative factors such as tea polyphenols and flavonoids exert inhibitory effect on chemical mediator release from rat peritoneal exudate cells (PEC). Some of these immunoregulatory activities of food components could be expressed in vivo. Oral administrations of perilla and fish oils or tea polyphenols led to a significant decrease of LTB4 releasing activity of rat PEC, but IgE production enhancing activity of PUFA was not expressed in vivo. On the other hand, alpha-tocopherol feeding enhanced serum IgA level as well as IgA productivity of mesenteric lymph node lymphocytes. These results suggest that dietary fats and antioxidants are effective for the alleviation of allergic symptom and activation of immune functions.

On the Role of Squalene in Olive Oil Stability

The role of squalene in olive oil stability was studied for various concentrations and experimental conditions. No effect was found in induction periods of olive oil at elevated temperatures using the Rancimat apparatus. Samples were then stored at 40 and 62 degrees C in the dark, and the extent of oxidation was followed by periodic measurements of peroxide value and conjugated dienes. A concentration dependent moderate antioxidant activity was evidenced which was stronger in the case of olive oil compared to that found for sunflower oil and lard. In the presence of alpha-tocopherol (100 mg/kg) and caffeic acid (10 mg/kg) the contribution of squalene (7000 mg/kg) was not significant. No radical scavenging activity was observed using DPPH(*) in 2-propanol. The weak antioxidant activity of squalene in olive oil may be explained by competitive oxidation of the different lipids present which leads to a reduction of the oxidation rate. Squalene plays a rather confined role in olive oil stability even at low temperatures.

Alpha-tocopherol decreases interleukin-1 beta release from activated human monocytes by inhibition of 5-lipoxygenase.

Cardiovascular disease is the leading cause of morbidity and mortality in westernized populations. Low levels of alpha-tocopherol (AT) are associated with increased incidence of atherosclerosis and increased intakes appear to be protective. Recently, we showed that supplementation with AT resulted in significant decreases in monocyte superoxide anion release, lipid oxidation, interleukin-1 beta (IL-1 beta) release, and adhesion to endothelium. The reduction in superoxide and lipid oxidation by AT seemed to be mediated by inhibition of protein kinase C. The aim of this study was to investigate the mechanism(s) by which AT inhibits IL-1 beta release. Potential mechanisms examined included its effect as an antioxidant and its inhibitory effects on protein kinase C and the cyclooxygenase-lipoxygenase pathways. Although AT decreased superoxide release from activated monocytes, superoxide dismutase and catalase had no effect on IL-1 beta release. Also, a similar antioxidant, beta-tocopherol, had no effect on IL-1 beta release. The protein kinase C inhibitor, bisindolylmaleimide, did not inhibit IL-1 beta release from activated monocytes, in spite of AT decreasing protein kinase C activity. Leukotriene B4, a major product of 5-lipoxygenase, has been shown to augment IL-1beta release. In the presence of AT, a significant reduction in leukotriene B4 and IL-1 beta levels was observed, which was reversed by the addition of leukotriene B4. Similar observations were seen with specific inhibitors of 5-lipoxygenase. The product of cyclooxygenase, prostaglandin E2, has been shown to inhibit IL-1 beta activity in some systems. However, AT had no significant effect on prostaglandin E2 levels in activated monocytes. In the presence of indomethacin, a cyclooxygenase inhibitor, AT inhibited IL-1 beta activity. Also, AT had no effect on IL-1 beta mRNA levels or stability, suggesting a posttranscriptional effect. Thus, in activated human monocytes, AT exerts a novel biological effect of inhibiting the release of the proinflammatory cytokine, IL-1 beta, via inhibition of the 5-lipoxygenase pathway.

Alpha-tocopherol inhibits oxidative stress induced by cholestanetriol and 25-hydroxycholesterol in porcine ovarian granulosa cells.

The cytotoxicity of oxysterols including 7-ketocholesterol, alpha-epoxide, cholestanetriol and 25-hydroxycholesterol and the possible protecting effect of alpha-tocopherol on cholestanetriol and 25-hydroxycholesterol-induced cytotoxicity were investigated in primary cultures of porcine ovarian granulosa cells. Cell viability as determined by % trypan blue staining and mitochondrial function as determined using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) reduction were decreased significantly after 24 h exposure to 2.5 microM alpha-epoxide, cholestanetriol and 25-hydroxycholesterol. 7-Ketocholesterol (2.5 microM) did not affect cell viability or mitochondrial function under the same culture conditions. The specific activities of catalase and superoxide dismutase, two antioxidant defense enzymes were increased significantly (p < 0.01) following 24 h exposure to 2.5 microM concentrations of cholestanetriol while only superoxide dismutase was increased in 25-hydroxycholesterol-treated cells (p < 0.001). Specific activity of glutathione peroxidase was unchanged relative to control cells. Levels of thiobarbituric acid reactive substances remained unchanged after exposure to 7-ketocholesterol, alpha-epoxide, cholestanetriol, 25-hydroxycholesterol and cholesterol. Administration of 1 microM alpha-tocopherol to the culture medium significantly improved cell viability and restored both superoxide dismutase and catalase activities to control levels in cholestanetriol -treated cells and only superoxide dismutase in 25-hydroxycholesterol-treated cells. These studies suggest that the cytotoxic nature of physiologically relevant concentrations of cholestanetriol and 25-hydroxycholesterol in granulosa cells is in part due to oxidative stress, but it may be reduced in the presence of alpha-tocopherol.

Molecular basis of alpha-tocopherol control of smooth muscle cell proliferation.

Rat and human vascular smooth muscle cell proliferation is specifically sensitive to alpha-tocopherol, but not beta-tocopherol. The former, but not the latter, is capable of limiting proliferation and inhibiting protein kinase C activity in a dose-dependent manner. The phenomenon occurs at concentrations in the range 10-50 microM. beta-tocopherol addition together with alpha-tocopherol, prevents both cell growth and protein kinase C inhibition. alpha-tocopherol increases de novo synthesis of protein kinase C molecules. The enzyme specific activity, however, is diminished, due to a decreased phosphorylation of protein kinase C, occurring in the presence of alpha-tocopherol. Experiments with protein kinase C isoform-specific inhibitors and precipitating antibodies show that the only isoform affected by alpha-tocopherol is protein kinase C-alpha. The effect of alpha-tocopherol is prevented by okadaic acid indicating a phosphatase of the PP2A type as responsible for protein kinase C-alpha dephosphorylation produced in the presence of alpha-tocopherol. At a gene level alpha-tocopherol but not beta-tocopherol induces a transient activation of alpha-tropomyosin gene transcription and protein expression. It is proposed that, by inhibiting protein kinase C activity via an activation of a phosphatase PP2A, alpha-tocopherol controls smooth muscle cell proliferation through changes in gene expression.

Antioxidant activity of carotenoids: an electron-spin resonance study on beta-carotene and lutein interaction with free radicals generated in a chemical system.

beta-Carotene is thought to be a chain-breaking antioxidant, even though we have no information about the mechanism of its antioxidant activity. Using electron-spin resonance (ESR) spectroscopy coupled to the spin-trapping technique, we have studied the effect of beta-carotene and lutein on the radical adducts of the spin-trap PBN (N-t-butyl-alpha-phenylnitrone) generated by the metal-ion breakdown of different tert-butyl hydroperoxide (tBOOH) concentrations in methylene chloride. The peroxyl radical, along with an oxidation product of PBN (the PBNOx), trapped at room temperature from the breakdown of high concentration of tBOOH (1 M), were quenched by beta-carotene or lutein, in competition with the spin-trapping agent. However, carotenoids were not able to quench the alkoxyl and methyl radicals generated in the reaction carried out in the presence of low tBOOH concentration (1 mM). The reaction between carotenoids and the peroxyl radical was also carried out in the absence of the spin trap, at 77 K: Under these different experimental conditions, we did not detect any radical species deriving from carotenoids. In the same system, a further evidence of the peroxyl radical quenching by beta-carotene and lutein was obtained. The antioxidant activity of vitamin E was also tested, for comparison with the carotenoids. In the presence of alpha-tocopherol, peroxyl and alkoxyl radicals were quenched, and the tocopheroxyl radical was detected. Our data provide the first direct evidence that carotenoids quench peroxyl radicals. Under our experimental conditions, we did not detect any carotenoid radical species that could derive from the interaction with the peroxyl radical. The radical-trapping activity of beta-carotene and lutein demonstrated in this chemical reaction contributes to our understanding carotenoid antioxidant action in biological systems.

Ascorbic acid 2-O-alpha-glucoside-induced redox modulation in human keratinocyte cell line, SCC: mechanisms of photoprotective effect against ultraviolet light B.

We previously reported that the topical application of ascorbic acid 2-O-alpha-glucoside (AA-2G) suppressed the cutaneous inflammation by ultraviolet irradiation in human and guinea pigs (Miyai et al., Nishinihon J. Dermatol., 58, 439-443 (1996)). In this paper, the effect of AA-2G on the lethal damage induced by ultraviolet B (UVB) was studied using a human keratinocyte cell line, SCC, established from squamous cell carcinoma. The photoprotective effect of AA-2G on cytotoxicity of UVB in SCC cells was dose dependent (0.125-1 mM) and more effective than that of ascorbic acid (AsA) at 1 mM. This protection was completely abolished in the presence of an alpha-glucosidase inhibitor, castanospermine, indicating that release of AsA from this derivative was essential for reduction of the actinic injury. AA-2G significantly suppressed cytotoxicities of hydrogen peroxide and superoxide anion produced by xanthine and xanthine oxidase. AA-2G exhibited a preventive effect against the cytotoxicity produced by tert-butylhydroperoxide, an inducer of lipid peroxidation, in the presence of alpha-tocopherol, but not in the absence of alpha-tocopherol. Cytotoxicity of UVB was also effectively reduced by the combination of AA-2G and alpha-tocopherol. In addition, AA-2G reduced UVB-promoted formation of lipid peroxide and accumulation of lipofuscin, which is known to be a complex of cellular proteins and metabolites of lipid peroxide. These data suggest that AA-2G prevents the acute inflammation induced by UVB irradiation partly through scavenging reactive oxygen species and potentiating the antioxidative activity of alpha-tocopherol.

Characteristics of the protective action of alpha-Tocopherol in vascular hypoxia.

The functioning of the guinea-pig isolated portal vein was monitored by measuring spontaneous mechanical activity, responses to electrical stimulation and administered noradrenaline in normoxic conditions. The effect of hypoxia, induced by bubbling the physiological bathing solution with a 95% N(2)/5% CO(2) gas mixture, on the mechanical performance of the vein was then assessed. Spontaneous activity declined in hypoxia, with mean contraction tension reduced by 55 + or - 8.8%. The responses to electrical field stimulation (2-32 Hz, 0.7 msec. 70 V) were lowered by 14 + or - 4.6% but contractions produced by a range of noradrenaline concentrations (0.01-160 mu M) were unaffected by hypoxia. Substitution of glucose in the bathing solution with sucrose, a substrate unavailable to the cells for energy generation, produced a marked enhancement of the effect of hypoxia. Spontaneous activity was reduced by 76 + or - 8.3%, electrically-induced activity by 80 + or - 14.4% and noradrenaline-induced responses by 85 + or - 6.8%. Although in normoxia the activity and responses of the portal vein were unaffected by the presence of alpha-tocopherol, it significantly protected the functioning of the vein in hypoxic conditions. This effect was concentration-dependent within the range 10-160 mu M and was most marked when glucose was replaced by sucrose in the bathing solution.

Effect of α‐tocopherol and Trolox on the decomposition of methyl linoleate hydroperoxides

To clarify the mechanisms of antioxidant action, the effect of alpha-tocopherol and its water-soluble carboxylic acid derivative, Trolox, was studied on the decomposition of methyl linoleate hydroperoxides (MeLoOOH). Decomposition rate and the distribution of autoxidation products formed from MeLoOOH were followed by analyzing the volatile and non-volatile products by static headspace gas chromatography and normal-phase high-performance liquid chromatography, respectively. Both alpha-tocopherol and Trolox markedly inhibited the decomposition of MeLoOOH in a concentration-dependent way. In the absence of antioxidants, MeLoOOH was completely decomposed after incubation for 48 h at 60 degrees C, and in the presence of equal molar concentration of antioxidants only 6-7% of initial MeLoOOH was decomposed even after 280 h of incubation. MeLoOOH produced 1.2% methyl linoleate hydroxy compounds (MeLoOOH) in the presence of alpha-tocopherol and 3.8% in the presence of Trolox. Both antioxidants inhibited the formation of volatile decomposition products and the formation of ketodiene compounds. The hydroxy compounds may be formed by the reaction of alkoxy radical and hydrogen donating antioxidants. Conversion of MeLoOOH into stable MeLoOH demonstrated that the antioxidants alpha-tocopherol and Trolox trap alkoxyl radicals by H-donation.

A novel approach to study linoleic acid autoxidation: importance of simultaneous detection of the substrate and its derivative oxidation products.

In this paper we have proposed a novel approach for studying the reaction of lipid oxidation by using the simplest chemical system available. Neat linoleic acid was incubated for 24 hours at 37 degrees C in the air. The course of lipid oxidation was followed by measuring simultaneously by HPLC with a diode array detector 1) linoleic acid decrease, 2) the products formed by radical attack, namely four hydroperoxy-octadeca-dienoic acid (HPODE) isomers, two c,t (c,t) and two trans,trans (t,t). 3) the byproducts formed by HPODE degradations, the four oxo-octadeca-dienoic acid (oxo-ODE) isomers. In HPODEs the presence of conjugated diene chromophore was confirmed by second derivative spectrophotometry. c,t HPODEs were also identified for their positional isomerism, while for t,t molecules the lack of suitable reference compound makes unfeasible the identification of their positional isomerism. As in the case of the latter two c,t and two t,t oxo-ODE isomers were characterized. This simple system appears to be useful for studying the activity exherted by lipophilic molecules that, like alpha-tocopherol, may act as antioxidants and/or as hydrogen atom donating molecules. The presence of alpha-tocopherol in different concentration for 24 hours in the reaction environment, shifts the reaction of linoleic acid autoxidation towards different byproduct formations. From the results obtained it is evident that alpha-tocopherol acts as hydrogen atom donor at all concentration tested, shifting the reaction toward a prevalent formation of c,t isomer of both HPODEs and oxo-ODEs. At concentration lower than 40 nmoles, when the ratio between alpha-tocopherol and linoleic acid was 1:100, the reaction of autoxidation is strongly inhibited, while at higher concentration alpha-tocopherol acted as a prooxidant. In these experimental conditions, alpha-tocopherylquinone was spectrophotometrically identified as the predominant oxidation product of alpha-tocopherol.

Oxidative Damage to Lens Membranes Induced by Metal-Catalyzed Systems

The oxidative effect of hydrogen peroxide, ascorbic acid and glucose in the presence of transition metals was studied. Incubation of bovine lens membranes with hydrogen peroxide/metal, ascorbic acid/metal and glucose/metal systems resulted in a significant augmentation of thiobarbituric acid-reactive substance content in the membranes. Presence of alpha-tocopherol decreased the extent of oxidative damage. This oxidation was found to be mediated by hydroxyl radicals. Dependence of hydroxyl radical generation upon the buffer used has been observed. In all experiments Cu(II) ions exhibited a higher efficiency compared to Fe(II) ions.

Characterisation of clusters of alpha-tocopherol in gel and fluid phases of dipalmitoylglycerophosphocholine.

The effect of alpha-tocopherol on the phase behaviour of aqueous dispersions of dipalmitoylglycerophosphocholine has been examined by differential scanning calorimetry, freeze-fracture electron microscopy, and real-time X-ray diffraction methods. The presence of alpha-tocopherol in proportions 2.5, 5, 7.5, and 10 mol/100 mol results in a progressive decrease in the temperature of the gel to liquid-crystalline phase transition from 41.5 degrees C to 36 degrees C and a reduction in transition enthalpy from 35 kJ.mol-1 to 15 kJ.mol-1 phospholipid. The thermal data indicated that the pretransition of the phospholipid is eliminated even in mixtures containing 2.5 mol/100 mol alpha-tocopherol. Real-time X-ray diffraction measurements using synchrotron radiation performed under identical conditions to the thermal studies showed clear transition sequences of L beta-->P beta-->L alpha for all mixtures. The sequence was reversible with hysteresis of 2-3 degrees C on cooling. Low-angle X-ray scattering from mixtures in the gel phase showed three lamellar repeat spacings of 6.35, 7.5, and 8.4 nm. The spacing at 6.35 nm was assigned to pure phospholipid from which alpha-tocopherol has been phase separated into enriched domains giving lamellar repeat spacings of 7.5 nm and 8.4 nm. Low-angle diffraction patterns of mixtures in the fluid phase were characterised by two lamellar repeat spacings. The longer spacing of about 6.6 nm was assigned to pure phospholipid and the shorter spacing at about 6.1 nm to an alpha-tocopherol-enriched phase. Electron microscopy of freeze-fracture replicas of mixtures of phospholipid containing 10 mol/100 mol alpha-tocopherol thermally quenched from 10 degrees C and 60 degrees C, showed evidence of domain structures within the bilayer plane that appeared to be correlated between successive bilayers in multilamellar dispersions. Calculations of the stoichiometry of phospholipid: alpha-tocopherol in the alpha-tocopherol-enriched domains based on enthalpy data and integrated X-ray scattering intensity gave values of 9.6:1 for the fluid phase and 9.2:1 for the gel phase. This was consistent with a clustering of alpha-tocopherol molecules in both gel and liquid-crystal phases of dipalmitoyl-glycerophospholcholine in approximately the same stoichiometry.

Sesamin (a compound from sesame oil) increases tocopherol levels in rats fed ad libitum.

Six groups of rats were fed diets low, but adequate, in alpha-tocopherol but high in gamma-tocopherol. The six diets differed only in their contents (0, 0.25, 0.5, 1.0, 2.0, and 4.0 g/kg, respectively) of sesamin, a lignan from sesame oil. After four weeks of ad libitum feeding, the rats were sacrificed and the concentrations of alpha- and gamma-tocopherols were measured in the plasma, livers, and lungs. Sesamin-feeding increased gamma-tocopherol and gamma-/alpha-tocopherol ratios in the plasma (P < 0.05), liver (P < 0.001), and lungs (P < 0.001). The increase was non-significant for alpha-tocopherol. Thus, sesamin appears to spare gamma-tocopherol in rat plasma and tissues, and this effect persists in the presence of alpha-tocopherol, a known competitor to gamma-tocopherol. This suggests that the bioavailability of gamma-tocopherol is enhanced in phenol-containing diets as compared with purified diets.

Chemilumineszenzmethode zum Nachweis strahleninduzierter Oxidationsprodukte in fetthaltigen Lebensmitteln

Radiation-induced oxidation of fatty foods was detected by a chemiluminescence method. Hazelnuts, peanuts and poultry were used as foodstuff samples. Additional investigations were performed with a model system and sunflower oil. The irradiation of the samples was carried out in a x-ray-fluorescence-apparatus. Thereby it is to note that the G-value of the x-ray-radiation is much higher than the G-value of a cobalt-60-source normally used for irradiation of food. A dependence of the integral of the light curve on the irradiation doses could be proved. Investigations with model systems which contained different amounts of alpha-tocopherol showed a decreasing chemiluminescence signal at low irradiation doses in presence of alpha-tocopherol. At higher doses the chemiluminescence signal enlarges with increasing amounts of alpha-tocopherol because irradiation products of alpha-tocopherol overlay its antioxidative effect. Irradiated poultry samples differ significantly from unirradiated samples after a deep-freeze storage of 26 weeks. A quantification of the doses is not possible without knowledge of the storage time, because the integrals decrease differently after irradiation during storage. In any case the chemiluminescence method is useful as a "screening method" for the detection of irradiation of foodstuffs with the possibility of automation and high sensitivity.

Free and albumin-bound bilirubin are efficient co-antioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation

Peroxidation of the lipid moieties of low density lipoproteins (LDL) is regarded as an early event in atherogenesis. Because bilirubin is a physiological reductant with antioxidant activities, we investigated its inhibitory action on the radical-mediated oxidation of LDL and plasma lipids. Exposing fresh human blood plasma to lipophilic peroxyl radicals generated from 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) resulted in rapid oxidation of ubiquinol-10, followed by that of ascorbate and bilirubin. Plasma lipids were well protected from peroxidation as long as these three antioxidants were present, as assessed by the amounts of cholesterylester hydroperoxides formed during this period. Following consumption of these antioxidants, and in the presence of alpha-tocopherol, the rate of hydroperoxide formation increased sharply with roughly 2 molecules of cholesterylester hydroperoxides being formed for each peroxidation initiating event. Supplementation of AMVN-oxidizing plasma with exogenous bilirubin at the onset of rapid lipid peroxidation, i.e. after depletion of endogenous ubiquinol-10, ascorbate, and bilirubin, led to a halt in both hydroperoxide formation and consumption of alpha-tocopherol. When isolated LDL was incubated with AMVN, approximately 9 molecules of cholesterylester hydroperoxides were formed per peroxidation initiating event and while alpha-tocopherol was consumed. Addition of free or albumin-bound bilirubin to isolated LDL at the onset of oxidation resulted in a strong inhibition of hydroperoxide formation and alpha-tocopherol consumption, the effect being more pronounced with the free pigment. Addition of the corresponding amounts of albumin alone was without effect. In the presence of albumin-bound bilirubin, some 30% of the pigment was initially converted into biliverdin, whereas formation of this oxidation product was not observed with the free pigment. Also, the presence of bilirubin oxidase partially reversed the inhibitory activity of bilirubin on AMVN-induced LDL oxidation in the absence but not presence of albumin. An attenuation of hydroperoxide formation and a temporary increase in LDL's alpha-tocopherol concentration were observed when free- or albumin-bound bilirubin were added to AMVN-oxidizing, alpha-tocopherol-containing LDL. In contrast, hydroperoxide formation was not inhibited significantly when the albumin-bound pigment was added to oxidizing LDL after complete consumption of its alpha-tocopherol. Our results show that bilirubin inhibits oxidation of LDL lipids initiated within the lipoprotein core and indicate that this activity is mediated by interaction of the pigment with LDL's alpha-tocopherol.

Emulsion multiple de type L/H/L: étude de l'obtention et du mécanisme de libération

Synopsis An O/W/O multiple emulsion was formulated using almond oil as the inner phase and liquid paraffin as the outer phase. The properties of the formulation ingredients were explored: effect of the type of primary emulsifier, and the nature and concentration of the secondary emulsifier. The volume fraction of the internal phase in the multiple emulsion was also optimized with regard to the stability. The multiple systems were assessed by evaluating several parameters such as the macroscopic aspect, the droplet size, the direction of the dispersion and the accelerated stability under centrifugation or elevated temperature. The effect of some additives (ozokerite, benzyl alcohol and glycerol) was examined. Whatever additive was used, a decrease in the formulation stability, which could be attributed to different phenomena, was observed. Furthermore, an analytical procedure was developed for studying the release of an active principle, alpha-tocopherol (vitamin E). This release could be a result of the instability of the multiple droplets or a diffusion through the water layer membrane. This procedure was based on the difference of chemical type between the two oil phases: mineral outer phase and fatty inner phase. Two chromatographic methods were used: a gas chromatographic assay of oleic acid which is the main component of the fatty phase, and a liquid chromatographic assay for the alpha-tocopherol, encapsulated in the inner phase. The presence of oleic acid in the paraffin phase originated from a rupture of the multiple droplets, whereas the presence of alpha-tocopherol in the outer phase could be due both to a diffusion phenomenon and to the instability of the formulation. The results obtained showed that only the rupture of the droplets occurred. Moreover, a part of the alpha-tocopherol was retained by Hypermer A 60 used as an emulsifier in the formulation.

The stability of β-carotene under different laboratory conditions

The stability of β-carotene, the carotenoid with the highest vitamin A activity, has been investigated under laboratory conditions, other than food processing or food storage, β-Carotene appears to be fairly stable over 24 h (loss< 4%) and 48 h (loss < 15%) when micellar solutions in aqueous medium of this pigment were incubated at 37° C, in the dark, in a 5% CO 2 in air atmosphere to simulate incubation conditions (used for cell culture systems). The effect of both UV light and fluorescent light on solutions of β-carotene in toluene was highly damaging (50% loss occurred after 8 h under UV light and after 24 h under daylight). Butylated hydroxytoluene (BHT) and alpha-tocopherol, two common antioxidants, were able to reduce significantly the degradation of β-carotene under light exposure. At the same concentration of BHT (1 m M), alpha-tocopherol had a much stronger relative potency to prevent the loss of the pigment (50% loss occurred after 48 h in fluorescent and 40 h in UV light in presence of alpha-tocopherol and after 23 h and 17 h, respectively, in fluorescent and UV light in presence of BHT). Increasing the rate of air-solution exchange by increasing the surface of β-carotene solution exposed to air caused an increase of the loss of the pigment of more than 3 times. Under storage conditions (−20° C, in the dark, under N 2), the rate of loss of β-carotene was 1.5%/month and 1.1%/month, respectively, in absence or in presence of 0.025% BHT.

Effect of alpha-tocopherol on the volatile thermal decomposition products of methyl linoleate hydroperoxides.

alpha-Tocopherol and 1,4-cyclohexadiene were tested for their effect on the thermal decomposition of methyl linoleate hydroperoxide isomers. The volatiles generated by thermolysis in the injector port of a gas chromatograph at 180 degrees C were analyzed by capillary gas chromatography. In the presence of either alpha-tocopherol or 1,4-cyclohexadiene, which are effective donors of hydrogen by radical abstraction, volatile formation decreased in all tests, and significant shifts were observed in the relative distribution of products in certain hydroperoxide samples. When an isomeric mixture of methyl linoleate hydroperoxides (cis, trans, and trans,trans 9- and 13-hydroperoxides) was decomposed by heat, the presence of alpha-tocopherol and 1,4-cyclohexadiene caused the relative amounts of pentane and methyl octanoate to decrease and hexanal and methyl 19-oxononanoate to increase. A similar effect of alpha-tocopherol was observed on the distribution of volatiles formed from a mixture of the trans,trans 9- and 13-hydroperoxides. This effect of alpha-tocopherol was, however, insignificant with pure cis,trans 13-hydroperoxide of methyl linoleate. The decrease in total volatiles with the hydrogen donor compounds, alpha-tocopherol and 1,4-cyclohexadiene, indicates a suppression of homolytic beta-scission of the hydroperoxides, resulting in a change in relative distribution of volatiles. The increase in hexanal and methyl 9-oxononanoate at the expense of pentane and methyl octanoate in the presence of hydrogen donor compounds supports the presence of a heat-catalyzed heterolytic cleavage (also known as Hock cleavage), which seems to mainly affect the trans,trans isomers of linoleate hydroperoxides.