Competitive carotenoid and cholesterol incorporation into liposomes: effects on membrane phase transition, fluidity, polarity and anisotropy

Pure 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) or mixed DPPC:1,2-dipalmitoyl phosphatidyletanolamine (DPPE):1,2-dipalmitoyl diphosphatidylserine (DPPS) (17:5:3) liposomes were incorporated with 5 mol% dietary carotenoids (beta-carotene, lutein and zeaxanthin) or with cholesterol (16 and 48 mol%) in the absence or presence of 15 mol% carotenoids, respectively. The carotenoid incorporation yields ranged from 0.42 in pure to 0.72 in mixed phospholipid liposomes. They decreased significantly, from 3 to 14%, in the corresponding cholesterol-doped liposomes, respectively. Highest incorporation yields were achieved by zeaxanthin and lutein in phospholipid liposomes while in cholesterol-containing liposomes, lutein was highest incorporated. The effects on membrane structure and dynamics were determined by differential scanning calorimetry, steady-state fluorescence and anisotropy measurements. Polar carotenoids and cholesterol cause similar, dose-dependent effects: ordering and rigidification revealed by broadening of the transition peak, and increase of anisotropy. Membrane hydrophobicity is determined by cholesterol content and carotenoid polarity. In cholesterol-doped liposomes, beta-carotene is less incorporated than in cholesterol-free liposomes. Our observations suggest effects of carotenoids, even at much lower effective concentrations than cholesterol (8 to 80-fold), on membrane structure and dynamics. Although they are minor constituents of animal membranes, carotenoids may act as modulators of membrane phase transition, fluidity, polarity and permeability, and therefore, can influence the membrane physiology and pathology.     

Different ways to insert carotenoids into liposomes affect structure and dynamics of the bilayer differently

We apply and quantify two techniques to incorporate carotenoids into liposomes: (i). preparation of unilamellar liposomes from mixtures of phospholipids and a carotenoid or cholesterol; (ii). insertion of carotenoids into prepared liposomes. Homogeneous liposomal fractions with a vesicle size diameter of approximately 50 nm were obtained by an extrusion method. The resulting vesicles were subjected to a three-dimensional light scattering cross-correlation measurement in order to evaluate their size distribution. The fluorescent dyes Laurdan, DiI-C(18), C(6)-NBD-PC were used to label the liposomes and to evaluate modulations of ordering, hydrophobicity and permeability to water molecules adjacent to the bilayer in the presence of carotenoids and/or cholesterol. Zeaxanthin incorporation (up to 0.1-1 mol%) attributes to the symmetric and ordered structure of the bilayer, causing both a strong hydrophobicity and a lower water permeability at the polar region of the membrane. The incorporation of lutein has similar effects, but its ordering effect is inferior in the polar region and superior in the non-polar region of the membrane. beta-Carotene, which can be incorporated at lower effective concentrations only, distributes in a more disordered way in the membrane, but locates preferentially in the non-polar region and, compared to lutein and zeaxanthin, it induces a less ordered structure, a higher hydrophobicity and a lower water permeability on the bilayer.     

Macular pigments lutein and zeaxanthin as blue light filters studied in liposomes.

Lutein and zeaxanthin are the predominant carotenoids in the human macula lutea. Epidemiological data suggest that an increased intake of a lutein-rich diet correlates with a diminished risk for age-related macular degeneration, a major cause of impaired vision in the elderly. Filtering of blue light has been proposed as a possible mechanism of protection. Here, the blue light filter efficacy of carotenoids was investigated in unilamellar liposomes loaded in the hydrophilic core space with a fluorescent dye, Lucifer yellow, excitable by blue light. Carotenoids were incorporated into the lipophilic membrane. Fluorescence emission in carotenoid-containing liposomes was lower than in carotenoid-free controls when exposed to blue light, indicating a filter effect. Filter efficacy was in the order lutein > zeaxanthin > beta-carotene > lycopene. Some of the difference in blue light filter efficacy of carotenoids is attributable to differences in extinction coefficients, and a major further contribution is suggested to be related to the orientation of the incorporated molecules in the liposomal membrane.     

Carotenol fatty acid esters: easy substrates for digestive enzymes?

To study the specificity of gastric lipases on carotenoid mono- and diesters, an enzymatic assay was applied. Digestions were carried out in phosphate buffer at pH 7.4 and 37 °C. As substrates we employed oleoresins from marigold (Tagetes erecta L.; lutein diesters), red paprika (Capsicum annuum L., mainly capsanthin diesters), papaya (Carica papaya L.; β-cryptoxanthin esters), and loquat (Eriobotrya japonica Lindl.; β-cryptoxanthin esters) as well as retinyl palmitate. These were reacted with porcine pancreatic lipase, porcine pancreatin, porcine cholesterol esterase, and human pancreatic lipase. As reference enzyme a yeast lipase from Candida rugosa was applied. A high turnover could be observed with porcine pancreatic lipase and porcine cholesterol esterase, indicating cholesterol esterase to be a plausible candidate for generation of free carotenoids in the gut. Human pancreatic lipase accepted only retinyl palmitate as substrate, carotenoid mono- and diesters were not hydrolyzed. The assay permits an approach for calculation of enzymatic activities towards carotenoid esters as substrates for the first time, which is based on the amount of enzyme formulation, present in the assay (U/mg solid). Furthermore, these studies provide deeper insight into carotenoid ester bioaccessibility.     

Time- and media-dependent secondary carotenoid accumulation in Haematococcus pluvialis

The green microalgae Haematococcus pluvialis synthesizes secondary carotenoids after exposure to environmental stress, a process that is used for the biotechnological production of astaxanthin (Ax). This study reports, for the first time, the medium-dependent changes in the carotenoid pattern throughout the cultivation process as well as the exact composition of carotenoids and their fatty acid mono- and diesters using LC-MS. Secondary carotenoid formation started immediately upon exposure to nutrient depletion and high light conditions. Ax and its corresponding mono- and diesters were detected simultaneously. After 15 days of cultivation, no significant changes were detected in carotenoid composition; however, the ratio between carotenoid mono- and diesters still varied. Main carotenoids were identified as Ax linolenate and Ax oleate, but also five adonirubin and one lutein monoester were detected. The influence of three different autotroph media was studied on carotenoid content, which reached a maximum 16.1 mg/g dry weight. The results indicate that media composition has an influence on the ratio of Ax mono- to diester but not on the qualitative composition of secondary carotenoids in H. pluvialis. Beside the pathway via echinenone, canthaxanthin and adonirubin the results indicate that Ax biosynthesis takes place via another route: from beta-carotene via beta-cryptoxanthin, zeaxanthin and adonixanthin.     

Carotenoid-containing unilamellar liposomes loaded with glutathione: a model to study hydrophobic-hydrophilic antioxidant interaction

Unilamellar liposomes are used as a simple two-compartment model to study the interaction of antioxidants. The vesicle membrane can be loaded with lipophilic compounds such as carotenoids or tocopherols, and the aqueous core space with hydrophilic substances like glutathione (GSH) or ascorbate, mimicking the interphase between an aqueous compartment of a cell and its surrounding membrane.

Unilamellar liposomes were used to investigate the interaction of GSH with the carotenoids lutein, β-carotene and lycopene in preventing lipid peroxidation. Lipid peroxidation was initiated with 2,2′-azo-bis-[2,4-dimethylvaleronitrile] (AMVN). Malondialdehyde (MDA) formation was measured as an indicator of oxidation; additionally, the loss of GSH was followed. In liposomes without added antioxidant, MDA levels of 119 ± 6 nmol/mg phospholipid were detected after incubation with AMVN for 2 h at 37°C. Considerably lower levels of 57 ± 8 nmol MDA/mg phospholipid were found when the liposomal vesicles had been loaded with GSH. Upon incorporation of β-carotene, lycopene or lutein, the resistance of unilamellar liposomes towards lipid peroxidation was further modified. An optimal further protection was observed with 0.02 nmol β-carotene/mg phospholipid or 0.06 nmol lycopene/mg phospholipid. At higher levels both these carotenoids exhibited prooxidant effects. Lutein inhibited lipid peroxidation in a dose-dependent manner between 0.02 and 2.6 nmol/mg phospholipid. With increasing levels of lycopene and lutein the consumption of encapsulated GSH decreased moderately, and high levels of β-carotene led to a more pronounced loss of GSH.

The data demonstrate that interactions between GSH and carotenoids may improve resistance of biological membranes towards lipid peroxidation. Different carotenoids exhibit specific properties, and the level for optimal protection varies between the carotenoids.     

The solubilisation pattern of lutein, zeaxanthin, canthaxanthin and beta-carotene differ characteristically in liposomes, liver microsomes and retinal epithelial cells

The incorporation efficiencies of lutein, zeaxanthin, canthaxanthin and beta-carotene into Retinal Pigment Epithelial (RPE) cells (the human RPE cell line D 407), liver microsomes and EYPC liposomes are investigated. In RPE cells the efficiency ratio of lutein and zeaxanthin compared to canthaxanthin and beta-carotene is higher than in the other membranes. The preferential interactions of lutein and zeaxanthin with RPE cells are discussed considering special protein binding properties. Incorporation yields were obtained from the UV-Vis spectra of the carotenoids. Membrane modulating effects of the carotenoids were obtained from the fluorescence spectra of co-incorporated Laurdan (6-dodecanoyl-2-dimethylaminonaphtalene). The Laurdan fluorescence quenching efficiencies of the membrane bound carotenoids offer an access to direct determinations of membrane carotenoid concentrations. Fetal calf serum as carrier for carotenoid incorporation appears superior to tetrahydrofuran.     

Carotenoid-containing unilamellar liposomes loaded with glutathione: a model to study hydrophobic-hydrophilic antioxidant interaction

Unilamellar liposomes are used as a simple two-compartment model to study the interaction of antioxidants. The vesicle membrane can be loaded with lipophilic compounds such as carotenoids or tocopherols, and the aqueous core space with hydrophilic substances like glutathione (GSH) or ascorbate, mimicking the interphase between an aqueous compartment of a cell and its surrounding membrane.

Unilamellar liposomes were used to investigate the interaction of GSH with the carotenoids lutein, β-carotene and lycopene in preventing lipid peroxidation. Lipid peroxidation was initiated with 2,2'-azo-bis-[2,4-dimethylvaleronitrile] (AMVN). Malondialdehyde (MDA) formation was measured as an indicator of oxidation; additionally, the loss of GSH was followed. In liposomes without added antioxidant, MDA levels of 119 ± 6 nmol/mg phospholipid were detected after incubation with AMVN for 2 h at 37°C. Considerably lower levels of 57 ± 8 nmol MDA/mg phospholipid were found when the liposomal vesicles had been loaded with GSH. Upon incorporation of β-carotene, lycopene or lutein, the resistance of unilamellar liposomes towards lipid peroxidation was further modified. An optimal further protection was observed with 0.02 nmol β-carotene/mg phospholipid or 0.06 nmol lycopene/mg phospholipid. At higher levels both these carotenoids exhibited prooxidant effects. Lutein inhibited lipid peroxidation in a dose-dependent manner between 0.02 and 2.6 nmol/mg phospholipid. With increasing levels of lycopene and lutein the consumption of encapsulated GSH decreased moderately, and high levels of β-carotene led to a more pronounced loss of GSH.

The data demonstrate that interactions between GSH and carotenoids may improve resistance of biological membranes towards lipid peroxidation. Different carotenoids exhibit specific properties, and the level for optimal protection varies between the carotenoids.     

Carotenoids are degraded by free radicals but do not affect lipid peroxidation in unilamellar liposomes under different oxygen tensions

It has been questioned whether carotenoids can act as antioxidants in biological membranes. Biological membranes can be modeled for studies of lipid peroxidation using unilamellar liposomes. Both carotenoid depletion and lipid peroxidation were increased with increasing oxygen tension in unilamellar liposomes. Carotenoids in such liposomes were found to be very sensitive to degradation by free radicals generated from iron and 2,2'-azobis(2-amidinopropane) dihydrochloride, but they were not protective against lipid peroxidation. Lycopene and beta-carotene were more sensitive to free radical attack than lutein, zeaxanthin, and beta-cryptoxanthin.     

Singlet oxygen quenching by dietary carotenoids in a model membrane environment

The ability of several dietary carotenoids to quench singlet oxygen in a model membrane system (unilamellar DPPC liposomes) has been investigated. Singlet oxygen was generated in both the aqueous and the lipid phase, with quenching by a particular carotenoid independent of the site of generation. However, singlet oxygen quenching is dependent on the carotenoid incorporated; xanthophylls exhibit a marked reduction in efficiency compared to the hydrocarbon carotenoids. Lycopene and beta-carotene exhibit the fastest singlet oxygen quenching rate constants (2.3-2.5 x 10(9)M(-1)s(-1)) with lutein the least efficient (1.1 x 10(8)M(-1)s(-1)). The other carotenoids, astaxanthin and canthaxanthin, are intermediate. Zeaxanthin exhibits anomalous behavior, and singlet oxygen quenching decreases with increasing amounts of zeaxanthin, leading to nonlinear plots for the decay of singlet oxygen with zeaxanthin concentration. Such differences are discussed in terms of carotenoid structure and their influence on the properties of the lipid membrane. The formation of aggregates by the polar carotenoids is also proposed to be of significance in their ability to quench singlet oxygen.     

Spin-labelled lutein as a new antioxidant in protection against lipid peroxidation

A new potentially antioxidant compound, spin-labelled lutein (SL-lut), was synthesized and incorporated into egg yolk phosphatidylcholine (EYPC) liposome membrane. The approximate location of nitroxide free radical groups of SL-lut was determined based on electron paramagnetic resonance (EPR) spectra. Then the ability of SL-lut to protect EYPC liposomes against lipid peroxidation (LPO) was compared to the antioxidant effects of lutein and a nitroxide spin label 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (3-CP). Two free radical generation systems were used—a thermal decomposition of 2,2'-azobis (2,4 dimethyl-valeronitrile) (AMVN) and a modified Fenton reaction using ferric-8-hydroxyquinoline (Fe(HQ)₃). Determination of the amount of thiobarbituric acid reactive species (TBARS) was used as a measure of LPO. SL-lut was the most powerful antioxidant, reducing LPO by about 6-times in AMVN-treated liposomes and 7-times in Fe(HQ)₃-treated liposomes. Lutein alone gave only a moderate protection in both systems, while 3-CP was as efficient as SL-lut in the presence of AMVN, but not efficient whatsoever in the presence of Fe(HQ)₃. The results suggest that a nitroxide part of SL-lut plays an important role in enhancing the antioxidant activity of lutein and makes SL-lut a powerful antioxidant efficient under different conditions.     

Spin-labelled lutein as a new antioxidant in protection against lipid peroxidation

A new potentially antioxidant compound, spin-labelled lutein (SL-lut), was synthesized and incorporated into egg yolk phosphatidylcholine (EYPC) liposome membrane. The approximate location of nitroxide free radical groups of SL-lut was determined based on electron paramagnetic resonance (EPR) spectra. Then the ability of SL-lut to protect EYPC liposomes against lipid peroxidation (LPO) was compared to the antioxidant effects of lutein and a nitroxide spin label 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (3-CP). Two free radical generation systems were used—a thermal decomposition of 2,2′-azobis (2,4 dimethyl-valeronitrile) (AMVN) and a modified Fenton reaction using ferric-8-hydroxyquinoline (Fe(HQ)₃). Determination of the amount of thiobarbituric acid reactive species (TBARS) was used as a measure of LPO. SL-lut was the most powerful antioxidant, reducing LPO by about 6-times in AMVN-treated liposomes and 7-times in Fe(HQ)₃-treated liposomes. Lutein alone gave only a moderate protection in both systems, while 3-CP was as efficient as SL-lut in the presence of AMVN, but not efficient whatsoever in the presence of Fe(HQ)₃. The results suggest that a nitroxide part of SL-lut plays an important role in enhancing the antioxidant activity of lutein and makes SL-lut a powerful antioxidant efficient under different conditions.     

Astaxanthin and peridinin inhibit oxidative damage in Fe(2+)-loaded liposomes: scavenging oxyradicals or changing membrane permeability?

Astaxanthin and peridinin, two typical carotenoids of marine microalgae, and lycopene were incorporated in phosphatidylcholine multilamellar liposomes and tested as inhibitors of lipid oxidation. Contrarily to peridinin results, astaxanthin strongly reduced lipid damage when the lipoperoxidation promoters-H(2)O(2), tert-butyl hydroperoxide (t-ButOOH) or ascorbate-and Fe(2+):EDTA were added simultaneously to the liposomes. In order to check if the antioxidant activity of carotenoids was also related to their effect on membrane permeability, the peroxidation processes were initiated by adding the promoters to Fe(2+)-loaded liposomes (encapsulated in the inner aqueous solution). Despite that the rigidifying effect of carotenoids in membranes was not directly measured here, peridinin probably has decreased membrane permeability to initiators (t-ButOOH > ascorbate > H(2)O(2)) since its incorporation limited oxidative damage on iron-liposomes. On the other hand, the antioxidant activity of astaxanthin in iron-containing vesicles might be derived from its known rigidifying effect and the inherent scavenging ability.     

Differential effects of carotenoids on lipid peroxidation due to membrane interactions: X-ray diffraction analysis

The biological benefits of certain carotenoids may be due to their potent antioxidant properties attributed to specific physico-chemical interactions with membranes. To test this hypothesis, we measured the effects of various carotenoids on rates of lipid peroxidation and correlated these findings with their membrane interactions, as determined by small angle X-ray diffraction approaches. The effects of the homochiral carotenoids (astaxanthin, zeaxanthin, lutein, beta-carotene, lycopene) on lipid hydroperoxide (LOOH) generation were evaluated in membranes enriched with polyunsaturated fatty acids. Apolar carotenoids, such as lycopene and beta-carotene, disordered the membrane bilayer and showed a potent pro-oxidant effect (>85% increase in LOOH levels) while astaxanthin preserved membrane structure and exhibited significant antioxidant activity (40% decrease in LOOH levels). These findings indicate distinct effects of carotenoids on lipid peroxidation due to membrane structure changes. These contrasting effects of carotenoids on lipid peroxidation may explain differences in their biological activity.     

Differential effects of carotenoids on lipid peroxidation due to membrane interactions: X-ray diffraction analysis

The biological benefits of certain carotenoids may be due to their potent antioxidant properties attributed to specific physico-chemical interactions with membranes. To test this hypothesis, we measured the effects of various carotenoids on rates of lipid peroxidation and correlated these findings with their membrane interactions, as determined by small angle X-ray diffraction approaches. The effects of the homochiral carotenoids (astaxanthin, zeaxanthin, lutein, β-carotene, lycopene) on lipid hydroperoxide (LOOH) generation were evaluated in membranes enriched with polyunsaturated fatty acids. Apolar carotenoids, such as lycopene and β-carotene, disordered the membrane bilayer and showed a potent pro-oxidant effect (> 85% increase in LOOH levels) while astaxanthin preserved membrane structure and exhibited significant antioxidant activity (40% decrease in LOOH levels). These findings indicate distinct effects of carotenoids on lipid peroxidation due to membrane structure changes. These contrasting effects of carotenoids on lipid peroxidation may explain differences in their biological activity.     

Lutein and zeaxanthin as protectors of lipid membranes against oxidative damage: the structural aspects.

Two main xanthophyll pigments are present in the membranes of macula lutea of the vision apparatus of primates, including humans: lutein and zeaxanthin. Protection against oxidative damage of the lipid matrix and screening against excess radiation are the most likely physiological functions of these xanthophyll pigments in macular membranes. A protective effect of lutein and zeaxanthin against oxidative damage of egg yolk lecithin liposomal membranes induced by exposure to UV radiation and incubation with 2, 2'-azobis(2-methypropionamidine)dihydrochloride, a water-soluble peroxidation initiator, was studied. Both lutein and zeaxanthin were found to protect lipid membranes against free radical attack with almost the same efficacy. The UV-induced lipid oxidation was also slowed down by lutein and zeaxanthin to a very similar rate in the initial stage of the experiments (5-15 min illumination) but zeaxanthin appeared to be a better photoprotector during the prolonged UV exposure. The decrease in time of a protective efficacy of lutein was attributed to the photooxidation of the carotenoid itself. Both lutein and zeaxanthin were found to slightly modify mechanical properties of the liposomes in a very similar fashion as concluded on the basis of H(1) NMR and diffractometric measurements of pure egg yolk membranes and membranes pigmented with the xanthophylls. Linear dichroism analysis of the mean orientation of the dipole transition moment of the xanthophylls incorporated to the lipid multibilayers revealed essentially different orientation of zeaxanthin and lutein in the membranes. Zeaxanthin was found to adopt roughly vertical orientation with respect to the plane of the membrane. The relatively large orientation angle between the transition dipole and the axis normal to the plane of the membrane found in the case of lutein (67 degrees in the case of 2 mol% lutein in EYPC membranes) was interpreted as a representation of the existence of two orthogonally oriented pools of lutein, one following the orientation of zeaxanthin and the second parallel with respect to the plane of the membrane. The differences in the protective efficacy of lutein and zeaxanthin in lipid membranes were attributed to a different organization of zeaxanthin-lipid and lutein-lipid membranes.     

Differential accumulation and pigmenting ability of dietary carotenoids in colorful finches

Many animals develop bright red, orange, or yellow carotenoid pigmentation that they use to attract mates. Colorful carotenoid pigments are acquired from the diet and are either directly incorporated as integumentary colorants or metabolized into other forms before deposition. Because animals often obtain several different carotenoids from plant and animal food sources, it is possible that these pigments are accumulated at different levels in the body and may play unique roles in shaping the ultimate color expression of individuals. We studied patterns of carotenoid accumulation and integumentary pigmentation in two colorful finch species--the American goldfinch (Carduelis tristis) and the zebra finch (Taeniopygia guttata). Both species acquire two main hydroxycarotenoids, lutein and zeaxanthin, from their seed diet but transform these into a series of metabolites that are used as colorful pigments in the plumage (goldfinches only) and beak (both species). We conducted a series of carotenoid-supplementation experiments to investigate the relative extent to which lutein and zeaxanthin are accumulated in blood and increase carotenoid coloration in feathers and bare parts. First, we supplemented the diets of both species with either lutein or zeaxanthin and measured plasma pigment status, feather carotenoid concentration (goldfinches only), and integumentary color. Zeaxanthin-supplemented males grew more colorful feathers and beaks than lutein-supplemented males, and in goldfinches incorporated a different ratio of carotenoids in feathers (favoring the accumulation of canary xanthophyll B). We also fed goldfinches different concentrations of a standard lutein-zeaxanthin mix and found that at physiologically normal and high concentrations, birds circulated proportionally more zeaxanthin over lutein than occurred in the diet. Collectively, these results demonstrate that zeaxanthin is preferentially accumulated in the body and serves as a more potent substrate for pigmentation than lutein in these finches.     

Antioxidant activity of palm oil carotenes in peroxyl radical-mediated peroxidation of phosphatidyl choline liposomes

The antioxidant efficacy of alpha-carotene and comparison with beta-carotene in multilamellar liposomes prepared from egg yolk phosphatidyl choline (EYPC) exposed to the lipid soluble 2,2'-azobis (2,4-dimethyl valeronitrile) (AMVN) was investigated. Lipid peroxidation was measured as thiobarbituric acid reacting substances (TBARS) at 532 nm or as hydroperoxide formation at 234 nm after separation of phosphatidyl choline hydroperoxide (PCOOH) by high-pressure liquid chromatography (HPLC). Lutein and zeaxanthin, the hydroxyl derivatives of alpha- and beta-carotenes, and the chain breaking antioxidant alpha-tocopherol were also included in the study. AMVN being a lipid soluble, non polar azo initiator penetrates into the hydrophobic interior of the phospholipid bilayer, forming peroxyl radicals which peroxidate the phospholipid leading to PCOOH accumulation. All the carotenoids tested at 1 mol% relative to EYPC significantly suppressed the formation of PCOOH compared to control samples. In this system, alpha-carotene retarded PCOOH formation better than beta-carotene. Similarly, lutein was a better antioxidant than is zeaxanthin. But lutein and zeaxanthin were more effective antioxidants than alpha- and beta-carotenes, respectively. After 1 h of incubation of the carotenoid with AMVN, alpha-, beta-carotene, lutein and zeaxanthin limited PCOOH formation by 77%, 68%, 85% and 82%, respectively, while alpha-tocopherol elicited 90% reduction. AMVN incubated with EYPC for 2 h induced the formation of TBARS compared to control (P < 0.001). alpha-Carotene significantly suppressed the TBARS formation by 78% whilst beta-carotene, lutein, zeaxanthin and alpha-tocopherol elicited 60%, 91% and 80% reductions, respectively. Increasing the concentration of the carotenoid > 1 mol% to EYPC did not significantly increase protection of the membrane against free radical attack. Our findings suggest that alpha-carotene is a better antioxidant than is beta-carotene in phosphatidyl choline vesicles. It may, therefore, be useful in limiting free radical mediated peroxidative damage against membrane phospholipids in vivo.     

Studies of the distribution of vitamin A as ester and alcohol and of carotenoids in plasma proteins of several species

The distribution of vitamin A ester, alcohol, and lutein was studied in fractions of chicken plasma proteins obtained by ammonium sulfate fractionation or dialysis, 6–8 hr. after oral administration of vitamin A and lutein in an oily medium. The losses of vitamin A and carotenoids during fractionation were considerable in some cases, losses of vitamin A being as large as 50% at times. Although this made the interpretation of the distribution between precipitate and supernatant difficult, the following conclusions seem to be possible. Vitamin A ester was found to be associated with the least soluble, and vitamin A alcohol and lutein with the more soluble protein fractions. No mono- or diesters of lutein could be demonstrated in the plasma.