Stabilization of liposomal membranes by thermozeaxanthins: carotenoid-glucoside esters.

Thermozeaxanthins (TZS) are novel carotenoid-glucoside esters existing in the cell membranes of the thermophilic bacterium, Thermus thermophilus. The effect of TZS on membrane permeability was studied by measuring the leakage of the fluorescent dye from calcein-entrapped large unilamellar liposomes (LUVs). The LUVs were composed of a small portion (0.2-1.0 mol%) of TZS and phosphatidylcholine (PC) of various length and saturation degree of hydrocarbon chains. Incorporation of TZS in egg PC LUVs stabilized the liposomes in the temperature range from 30 to 80 degrees C, as only 2.6% of the entrapped calcein leaked out in contrast to 10% release from the egg PC liposomes without TZS. LUVs composed of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) were stabilized by the incorporation of TZS at a temperature below 30 degrees C. Inclusion of TZS in LUVs composed of dimyristoylphosphatidylcholine, whose hydrocarbon chains are shorter than both DPPC and DOPC, did not stabilize the liposomes. About 90% of the entrapped dye was lost indicating defects of the liposomal membranes. Matching of the lipid bilayer thickness with the molecular length of TZS in the bilayers is discussed.     

Interaction of retinol and retinoic acid with phospholipid membranes. A differential scanning calorimetry study.

The influence of retinol and retinoic acid, two retinoids of major interest, on the main gel to liquid-crystalline phase transition of different phospholipid membranes has been studied by means of differential scanning calorimetry. Both compounds exerted perturbing effects on the phase transition of membranes composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine. At concentrations up to 42.5 mol% of retinoid in the membrane, the delta H was not much affected with respect to the pure phospholipid, indicating a rather slight interaction. As the concentration of retinol was increased the Tc transition temperature decreased. A fluid-phase immiscibility was observed for the system DPPC/retinol at concentrations between 0 and 33 mol%. Almost ideal phase diagrams were obtained for the mixture DPPE/retinol. At concentrations of 33 mol% and higher retinol was able to induce phase separations in DPPC membranes, but not in DPPE. The effect of retinoic acid was much weaker, the Tc and delta H remaining almost unaltered and equal to that of the pure phospholipid up to concentrations of 30 mol%, at neutral pH. Retinoic acid exerted a pH-dependent effect. As the pH decreased, and therefore increased the extent of protonation of retinoic acid, the pertubation of the membrane induced by this compound was less. A strong effect, both on Tc and delta H, was observed at pH 10, where the retinoic acid moiety will be mainly unprotonated and the negative charge will generate repulsive forces thus destabilizing the membrane. The mixture DPPC/retinoic acid presents a region of fluid-phase immiscibility. At low pH, when the retinoic acid moiety was fully protonated, this fluid-immiscibility region extended from 0 to 36 mol% of retinoic acid, but its size decreased with increasing pH, and at pH 10 it was only found from 0 to 3 mol%. These results are discussed in terms of the possible retinoid/phospholipid interactions and the disposition of the retinoid moiety in the bilayer.     

Kinetin increases water permeability of phosphatidylcholine lipid bilayers

Kinetin is shown to increase substantially the water permeability of liposomes composed of several types of phosphatidylcholines including the natural phospholipids egg lecithin and asolectin and the synthetic phospholipids dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine. Kinetin effects were measured from 16.3 micromolar to 2.4 millimolar at temperatures from 10°C to 50°C and at pH 2.0, 7.0, and 11.0. Temperature studies indicate that kinetin produces a larger increase in water permeability with membranes in the more fluid liquid crystalline state. Kinetin is also shown to enhance [¹⁴C]glucose permeability and perhaps promotes membrane aggregation. From these experiments, we conclude that kinetin may produce its initial effect by altering the lipid bilayer portion of membranes.     

Relation between the prolongation of the anti-inflammatory activity of liposome-encapsulated hydrocortisone and liposome composition in experimental arthritis

Experiments on rabbits with arthritis have demonstrated the possibility of a 10-fold decrease in the dose of hydrocortisone acetate incorporated into liposomes, administered intraarticularly as compared with a commercial drug in the form of suspension. The antiinflammatory effect was found to be appreciably prolonged (up to 5-6 days) upon the use of dipalmitoylphosphatidylcholine liposomes with 20 mol% cholesterol. Hydrocortisone had a prolongation effect (about 1-2 days) in the lipid phase of multilamellar liposomes from egg lecithin, dimyristoylphosphatidylcholine and distearoylphosphatidylcholine.     

Cytochemical study of liposome and lipid vesicle phagocytosis

Electron microscopy cytochemistry has been used to study the cytoplasmic location of liposomes and lipid vesicles following specific antibody-dependent phagocytosis. The vesicle compositions were 94–99 mol% ‘fluid’ lipid (egg phosphatidylcholine or dimyristoylphosphatidylcholine at 37°C or ‘solid’ lipid (dipalmitoylphosphatidylcholine at 37°C). In some cases, 4 mol% phosphatidylserine was included in the vesicle membrane so as to vary the surface charge density. These vesicles undergo specific antibody-dependent phagocytosis by RAW264 macrophages when the lipid membranes contain 1–2 mol% dinitrophenyl lipid hapten in the presence of rabbit anti-dinitrophenyl IgG antibody. Internalized lipid vesicles can be visualized with the electron microscope when ferritin is trapped in the internal aqueous compartments prior to internalization. The lipid vesicles were demonstrated to be internal to the macrophage plasma membranes by selectively staining the plasma membranes with Ruthenium red. The cytoplasmic location of vesicles and liposomes was studied by electron microscopic staining for activities of the following enzymes: (1) acid phosphatase; (2) inorganic trimetaphosphatase; (3) adenosine triphosphatase; and (4) glucose-6-phosphatase. The first two enzymatic activities were found in association with ferritin-containing vesicles after antibody-dependent phagocytosis, showing the formation of vesicle-containing phagolysosomes. Adenosine triphosphatase and glucose-6-phosphatase were primary not associated with the vesicles, suggesting a minimal association of vesicles with plasma membrane, Golgi, endoplasmic reticulum and perinuclear cisternae. Phagosome-lysosome fusion did not appear to depend on the type of target lipid vesicle or liposome, on the ‘fluidity’ of the target membrane, or the presence of phosphatidylserine in the target membrane.     

Retinol metabolism in LLC-PK1 Cells. Characterization of retinoic acid synthesis by an established mammalian cell line

Specific assays, based on gas chromatography-mass spectrometry and high-performance liquid chromatography, were used to quantify the conversion of retinol and retinal into retinoic acid by the pig kidney cell line LLC-PK1. Retinoic acid synthesis was linear for 2-4 h as well as with graded amounts of either substrate to at least 50 microM. Retinoic acid concentrations increased through 6-8 h, but decreased thereafter because of substrate depletion (t1/2 of retinol = 13 h) and product metabolism (1/2 = 2.3 h). Retinoic acid metabolism was accelerated by treating cells with 100 nM retinoic acid for 10 h (t1/2 = 1.7 h) and was inhibited by the antimycotic imidazole ketoconazole. Feedback inhibition was not indicated since retinoic acid up to 100 nM did not inhibit its own synthesis. Retinol dehydrogenation was rate-limiting. The reduction and dehydrogenation of retinal were 4-8-fold and 30-60-fold faster, respectively. Greater than 95% of retinol was converted into metabolites other than retinoic acid, whereas the major metabolite of retinal was retinoic acid. The synthetic retinoid 13-cis-N-ethylretinamide inhibited retinoic acid synthesis, but 4-hydroxylphenylretinamide did not. 4'-(9-Acridinylamino)methanesulfon-m-anisidide, an inhibitor of aldehyde oxidase, and ethanol did not inhibit retinoic acid synthesis. 4-Methylpyrazole was a weak inhibitor: disulfiram was a potent inhibitor. These data indicate that retinol dehydrogenase is a sulfhydryl group-dependent enzyme, distinct from ethanol dehydrogenase. Homogenates of LLC-PK1 cells converted retinol into retinoic acid and retinyl palmitate and hydrolyzed retinyl palmitate. This report suggests that substrate availability, relative to enzyme activity/amount, is a primary determinant of the rate of retinoic acid synthesis, identifies inhibitors of retinoic acid synthesis, and places retinoic acid synthesis into perspective with several other known pathways of retinoid metabolism.     

Membrane ordering effects of the anticancer agent VM-26

The effect of the anticancer agent VM-26 on acyl chain order of cellular and model membranes was examined by electron spin resonance techniques. The order parameter for the paramagnetic probe 5-doxyl stearate was increased when VM-26 was incorporated into the bilayer of fluid-phase dimyristoylphosphatidylcholine (DMPC) or gel-phase dipalmitoylphosphatidylcholine (DPPC) liposomes at concentrations up to 4.8 mol%. The ordering effect of VM-26 in DMPC was greater than that of cholesterol on an equimolar basis. The less cytotoxic congener of VM-26, VP-16, was only one-third as active as VM-26 in its ordering effects on DMPC. Higher order parameters for 5-doxyl stearate were also noted in asolectin liposomes, Ehrlich ascites tumor cells, and CCRF-CEM cells treated with VM-26. We conclude that VM-26 has significant membrane associated activity in addition to its previously recognized nuclear effects.     

Phospholipid dependence of calcium ion effects on electrophoretic mobilities of liposomes

Electrophoretic light scattering (ELS) and depolarization of fluorescence have been used to determine the effect of membrane fluidity on the binding of Ca2+ to liposomes. ELS was used to measure the electrophoretic mobilities of the liposomes. Fluorescence depolarization was used to determine membrane fluidity. Zero to 30 mol% phosphatidylserine (PS) was incorporated into liposomes containing, as bulk phospholipids, one of the following: dimyristoyl-phosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), or hydrogenated egg phosphatidylcholine (H egg PC). The binding of Ca2+ to the liposomes appears to be influenced by membrane fluidity. Liposomes containing bulk phospholipids whose phase transition temperature is higher than the experimental temperature exhibit enhanced binding of CA2+.     

Comparison of the effects of inserted C40- and C50-terminally dihydroxylated carotenoids on the mechanical properties of various phospholipid vesicles

We have measured the extent of incorporation of zeaxanthin (C40) and decaprenozeaxanthin (C50) in unilamellar vesicles of dimyristoylphosphatidylcholine (n-C14) and dipalmitoylphosphatidylcholine (n-C16). The incorporation is larger when the molecular length of the carotenoid corresponds to the thickness of the phospholipid bilayer. Stereochemically pure 2,3-di-O-phytanyl-sn-glycero-1-phosphocholine was prepared by modification of the polar heads of the phospholipids of Halobacterium halobium. Vesicles of this branched-chain ether phospholipid incorporate poorly the carotenoids, whereas egg lecithin vesicles incorporate them better. Osmotic swelling and water permeability of vesicles, with or without carotenoids, were measured in a stopped-flow, light-scattering system. The reinforcing effect (lower permeability and higher rigidity) of carotenoids at 1.5 mol% incorporation into diphytanylphosphatidylcholine vesicles is comparable to that of 5 mol% cholesterol; however, carotenoids have no measurable effect on the egg lecithin vesicles. These results imply that the reinforcement of the membrane depends on a subtle adjustment of the phospholipid-carotenoid system.     

Interaction of wasp venom mastoparan with biomembranes

Mastoparan-induced changes in the K+ permeability of rat peritoneal mast cells, human erythrocytes, Staphylococcus aureus and Escherichia coli were examined. Mastoparan did not efficiently increase the K+ permeability of cells except for S. aureus. The release of membrane phospholipids was also observed from S. aureus cells in the concentration range of the permeability enhancement. Mastoparan stimulated histamine release from mast cells, independently of a small efflux of K+. Mastoparan became markedly effective to E. coli cells whose outer membrane structure was chemically disrupted beforehand, showing that the peptide can enhance the permeability of the cytoplasmic membranes of both Gram-positive and -negative bacteria. In experiments using liposomes, mastoparan increased the permeability of the liposomes composed of egg phosphatidylethanolamine and egg phosphatidylglycerol, which are the lipid constituents of the cytoplasmic membrane of E. coli cells, while it showed a weak activity to the liposomes composed of egg phosphatidylcholine and cholesterol. The latter result related closely to the fact that this peptide acted weakly on erythrocytes and mast cells in which acidic lipids constitute a minor portion. Mastoparan decreased the phase transition temperature of dipalmitoylphosphatidylglycerol liposomes, but it did not affect that of dipalmitoylphosphatidylcholine liposomes. These results indicate that mastoparan penetrated into membranes mainly containing acidic phospholipids and disrupted the membrane structure to increase the permeability. The action of the wasp venom mastoparan was compared with that of a bee venom melittin.     

Transfer properties of the bovine brain phospholipid transfer protein. Effect of charged phospholipids and of phosphatidylcholine fatty acid composition

The monolayer technique has been used to study the transfer of [¹⁴C]phosphatidylinositol from the monolayer to phosphatidylcholine vesicles. An equivalent transfer rate was found for egg phosphatidylcholine, dioleoylphosphatidylcholine, dielaidoylphosphatidylcholine and dipalmitoylphosphatidylcholine. A reduced transfer rate was found for a shorter-chain derivative, dimyristoylphosphatidylcholine, and for species with two polyunsaturated fatty acid chains such as dilinoleoylphosphatidylcholine, diheptadecadienoylphosphatidylcholine, dilinolenoylphosphatidylcholine and diether and dialkyl derivatives. No activity was found for 1,3-dipalmitoylphosphatidylcholine. The presence of up to 5 mol% phosphatidylinositol in egg phosphatidylcholine vesicles had no effect on the transfer rate. Introduction of more than 5 mol% phosphatidylinositol or phosphatidic acid into the phosphatidylcholine vesicles gradually decreased the rate of phosphatidylinositol transfer from the monolayer. 20 mol% acidic phospholipid was nearly completely inhibitory. Transfer experiments between separate monolayers of phosphatidylcholine and phosphatidylinositol showed that the protein-bound phosphatidylcholine is readily exchanged for phosphatidylinositol, but the protein-bound phosphatidylinositol exchange for phosphatidylcholine occurs at a 20-times lower rate. The release of phosphatidylinositol is dependent on the lipid composition and the concentration of charged lipid in the acceptor membrane, but also on the ratio between donor and acceptor membranes. The main transfer protein from bovine brain which transfer phosphatidylinositol and phosphatidylcholine transfers also phosphatidylglycerol, but not phosphatidylserine or phosphatidic acid. The absence of significant changes in the surface pressure indicate that the phosphatidylinositol and phosphatidylcholine transfer is not accompanied by net mass transfer.     

Binding of CTP: phosphocholine cytidylyltransferase to large unilamellar vesicles

We have studied the binding of CTP: phosphocholine cytidylyltransferase from HeLa cell cytosol to large unilamellar vesicles of egg phosphatidylcholine (PC) or HeLa cell phospholipids that contain various amounts of oleic acid. A fatty acid/phospholipid molar ratio exceeding 10% was required for CTP: phosphocholine cytidylyltransferase binding to liposomes. At a fatty acid/phospholipid molar ratio of 1; 85% of the cytosolic CTP: phosphocholine cytidylyltransferase was bound. The enzyme also bound to liposomes with at least 20 mol% palmitic acid, monoolein, diolein or oleoylacetylglycerol. Oleoyl-CoA did not promote enzyme binding to liposomes. Binding to oleate-PC vesicles was blocked by Triton X-100 but not by 1 M KCl, and was reversed by incubation of the vesicles with bovine serum albumin. Cytidylyltransferase bound to egg PC vesicles that contained 33 mol% oleic acid equally well at 4 degrees C and 37 degrees C. The enzyme also bound to dimyristoyl- and dipalmitoylphosphatidylcholine vesicles containing oleic acid at temperatures below the phase transition for these liposomes. Binding of the cytidylyltransferase to egg PC vesicles containing oleic acid, monoolein, oleoylacetylglycerol or diolein resulted in enzyme activation, as did binding to dipalmitoylPC-oleic acid vesicles. However, binding to egg PC-palmitic acid vesicles did not fully activate the transferase. Various mechanisms for cytidylyltransferase interaction with membranes are discussed.     

Vitamin E: inhibition of retinol-induced hemolysis and membrane-stabilizing behavior.

For the elucidation of the mechanism of membrane stabilization by vitamin E, the effects of alpha-tocopherol and its model compounds on either retinol-induced hemolysis of rabbit erythrocytes or the permeability and fluidity of liposomal membranes have been studied. Retinol-induced rabbit erythrocyte hemolysis has been found not to be caused by the oxidative disruption of erythrocyte membrane lipids initiated by retinol oxidation, but rather to arise from physical damage of the membrane micelle induced by penetration of retinol molecules. In suppressing hemolysis, alpha-tocopherol was more effective than other naturally occurring tocopherols. alpha-Tocopheryl acetate, nicotinate, and 6-deoxy-alpha-tocopherol were more effective than alpha-tocopherol itself. The inhibitory effects of alpha-tocopherol model compounds having side chains with at least two isoprene units or a long straight chain instead of the isoprenoid side chain were similar to those of alpha-tocopherol. These data suggest that for protection of membranes against retinol-induced damage, the hydroxyl group of alpha-tocopherol is not critical, but rather the chroman ring, three methyl groups on the aromatic ring, and the long side chain are necessary. To verify the mechanism of the inhibitory effect on hemolysis, not only the effect of vitamin E and its model compounds on the membrane permeability and fluidity, but also the mobility of alpha-tocopherol molecule in membranes has been investigated using bilayer liposomes as the model membranes. Addition of alpha-tocopherol to membranes produced a greater decrease in the permeability and fluidity of rat liver phosphatidylcholine liposomes compared with egg yolk phosphatidylcholine liposomes. In dipalmitoylphosphatidylcholine liposomes, however, alpha-tocopherol was less effective, that is, the more unsaturated the lipids, the more they interact with alpha-tocopherol. 2,2,5,7,8-Pentamethyl-6-chromanol with no isoprenoid side chain and phytol without the chromanol moiety had no effect. The measurement of 13C NMR relaxation times revealed that the mobility of methyl groups on the aromatic ring of alpha-tocopherol in membranes is significantly restricted. In contrast, the methyl groups at positions 4'a and 8'a on the isoprenoid side chain have high degrees of motional freedom in the lipid core of membranes. Furthermore, it was found that alpha-tocopherol in membranes interacts with chromate ions added as potassium chromate outside the membranes, resulting in an increase in membrane fluidity. These results are compatible with those of the inhibitory effect on retinol-induced erythrocyte hemolysis. On the basis of the results obtained here, a possible mechanism for membrane stabilization by vitamin E is proposed.     

Comparison of the effects of vitamin A and its analogs upon rabbit ear cartilage in organ culture and upon growth of the vitamin A-deficient rat

A study was conducted to explore the relationship between the effects of vitamin A upon cartilage and the biological role of vitamin A in maintaining growth and life. Retinol, retinoic acid, alpha-retinoic acid, and ROB-7699 (a cyclopentyl analog of retinoic acid) were highly effective in promoting the lysis of the extracellular matrix of cartilage grown in organ culture in vitro. Retinoic acid and its two analogs were quantitatively more active than was retinol in bringing about lysis of matrix and release of proteoglycan into the culture medium. A bioassay was then conducted to determine the ability of each compound to promote growth of vitamin A-deficient rats. In contrast to their effects upon cartilage, retinoic acid and its two analogs were considerably less active quantitatively than retinol in promoting growth of vitamin A-deficient rats. Moreover, the three acids tested showed graded biological activity in the growth bioassay, with alpha-retinoic acid showing reduced bioactivity (approx. one-fourth that of retinoic acid) and ROB-7699 being virtually inactive. The lysis of cartilage produced by these compounds was presumably caused by release of lysosomal enzymes as a result of the membrane-labilizing effects of the compounds. Thus, these membrane effects of the vitamin A-related compounds are poorly correlated with their biological growth-promoting activity. The alpha-ionone analogs of retinol and retinoic acid were able to maintain good health and growth of vitamin A-deficient rats, although their quantitative activity was low. Rats fed alpha-retinyl acetate showed high liver stores of alpha-retinyl esters and low levels of serum retinol-binding protein (similar to the levels seen in retinoic acid-fed rats). The biological activity of the alpha-ionone analogs was apparently not due to contamination with or conversion to the normal beta-ionone compounds.     

Separable binding proteins for retinoic acid and retinol in bovine retina

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [³H] retinoic acid which could not be effectively displayed by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [³H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

Retinol transfer across and between phospholipid bilayer membranes

The transfer of retinol across and between bilayer membranes was studied in vitro using unilamellar liposomes and erythrocytes. Transmembrane movement of retinol in phospholipid bilayer membranes was a spontaneous and rapid process with a halflife of less than 30 s. Retinol transfer between liposomes and between liposomes and erythrocytes was also a spontaneous and rapid process with a halflife of less than 10 min. The results suggest that retinol transport in the cell might not need the participation of specific transfer proteins.     

Delivery of liposome membrane-associated sterols through silastic membranes

The transport of sterols incorporated into the lecithin bilayer of small unilamellar liposomes through a model membrane was studied. A two-chamber diffusion cell containing liposomes with incorporated [4-¹⁴C]cholesterol or β-[4-¹⁴C]sitosterol in the donor chamber and liposomes with unlabeled cholesterol in the receiver chamber was used. The permeability coefficients of the sterols through silastic rubber membranes which served as a model membrane were measured. The permeability for cholesterol incorporated into liposomes in a phosphatidyl choline/cholesterol molar ratio of 1 : 1, produced by sonication for 1 h, and subsequent centrifugation at 100000 × g for 1 h, was 1.6 · 10^?8 cm sec^?1. Dilution of the liposome suspension did not change the permeability coefficient significantly. The permeability coefficient of sitosterol incorporated into liposomes was about 4-times smaller than that of cholesterol. These results suggest that the sterols were delivered to the silastic membrane by the intact liposomes and that free solute was not involved in the transport to the membrane to a significant degree. The large differences in the permeability coefficients between cholesterol and sitosterol indicate that an aqueous interfacial barrier was crossed by the sterol during the delivery to the membrane.     

Reaction of vitamin A with acceptors of electrons. Formation of radical anions from 7,7,8,8-tetracyanoquinodimethane and tetrachloro-1,4-benzoquinone

1. The interactions of retinol and retinoic acid with two electron acceptors, 7,7,8,8-tetracyanoquinodimethane (TCNQ) and tetrachloro-1,4-benzoquinone (chloranil), were studied in an investigation on the ability of vitamin A to behave as a donor of electrons. 2. Retinol reacts with TCNQ in polar organic solvents with the formation, as judged by spectral studies, of the radical anion of TCNQ. 3. Addition of the products of this reaction to water is accompanied by a rapid consumption of OH(-) ions. 4. Consumption of OH(-) ions is also a feature of the reactions between retinol and chloranil, but the spectrum of the radical anion of chloranil is observed only when retinol and chloranil are suspended in aqueous salt solutions. 5. Retinoic acid behaves similarly to retinol in its reactions with TCNQ and chloranil, but it appears to be a weaker electron donor than retinol. 6. The reaction products that may be formed from retinol in its reactions with TCNQ and chloranil are discussed. 7. It is suggested that the ability of vitamin A to behave as a donor of electrons may be an important aspect of its biochemical mode of action.     

The biosynthesis of retinoic acid from retinol by rat tissues in vitro

This report shows that a spectrum of vitamin A-dependent tissues can produce retinoic acid by synthesis in situ, indicates that cellular retinol and retinoic acid binding proteins are not obligatory to retinoic acid synthesis, and provides initial characterization of retinoic acid synthesis by rat tissues. Retinoic acid synthesis from retinol was detected in homogenates of rat testes, liver, lung, kidney, and small intestinal mucosa, but not spleen. Zinc did not stimulate the conversion of retinol into retinoic acid by liver homogenates. Retinoic acid synthesis was localized in cytosol of liver and kidney, where its rate of synthesis from retinol was fourfold (liver) and sevenfold (kidney) slower than from retinal. The synthesis of retinoic acid from retinol required NAD and was not supported by NADP. NADH (0.5 mM) reduced retinoic acid synthesis from retinol, supported by NAD (2 mM), by 50-70%, but was fivefold less potent in reducing retinoic acid synthesis from retinal. Dithiothreitol enhanced the conversion of retinol, but not retinal, into retinoic acid. EDTA inhibited the conversion of retinol into retinoic acid slightly (13%, liver; 29%, kidney). A high ethanol concentration (100 mM), relative to retinoid substrate (10 microM), inhibited retinoic acid synthesis from retinol (liver, 54%; kidney, 30%) and from retinal (30%, liver; 9%, kidney). 4'-(9-Acridinylamino)methansulfon-m-anisidine, an inhibitor of aldehyde oxidase, and disulfiram, a sulfhydryl-group crosslinking agent, were potent inhibitors of retinoic acid synthesis at 10 microM or less, and seemed equipotent in liver and kidney. 4-Methylpyrazole, an inhibitor of ethanol metabolism, also inhibited retinoic acid synthesis from retinol, but was less potent than the former two inhibitors, and affected liver to a greater extent than kidney, particularly with retinal as substrate.     

The effect of carotenoids on the concentration of singlet oxygen in lipid membranes

An effect of β-carotene and its polar derivative, zeaxanthin, on a concentration of singlet oxygen in lipid membranes was studied in a model system. The carotenoids were incorporated into the membranes of small unilamellar liposomes at a concentration of 0.15 mol% with respect to lipid. Singlet oxygen was generated in a liposome suspension via photosensitization of toluidine blue, and its concentration in a membrane was detected with application of a specific fluorescence probe (singlet oxygen sensor green reagent) located in the lipid bilayer. The results show the carotenoid-dependent decrease in the concentration of singlet oxygen in the membranes formed with unsaturated lipids (egg yolk phosphatidylcholine and digalactosyldiacylglycerol) but not in the case of the membranes formed with a saturated lipid (dimyristoylphosphatidylcholine). The effect of carotenoids was about twice as high as in the case of cholesterol present in liposomes at the same concentration. The results suggest that carotenoids protect membranes formed with unsaturated lipids against singlet oxygen through combined activity of different mechanisms: modification of structural properties of the lipid bilayers, physical quenching of singlet oxygen and chemical reactions leading to the pigment oxidation. The latter conclusion is based on the analysis of the absorption spectra of liposomes before and after light exposure. An importance of the different modes of protection by carotenoids against single oxygen toxicity towards biomembranes is discussed.