Vitamin D metabolites stimulate phosphatidylcholine transfer to renal brush-border membranes

The phosphatidylcholine content of both the intestinal and renal brush-border membranes and ion transport are affected by 1,25-dihydroxycholecalciferol (1,25(OH)2D3). To investigate the mechanism of this effect, liposomes were prepared containing self-quenching concentrations of fluorescent phospholipid derivatives. When these liposomes were incubated with rat renal brush-border membrane vesicles, an immediate increase in the relative fluorescence of N-4-nitrobenz-2-oxa-1,3-diazole phosphatidylcholine (NBD-PC) was detected, indicating transfer of NBD-PC into a non-quenched membrane. Addition of 1,25(OH)2D3 to the liposomes produced a dose-dependent stimulation of NBD-PC transfer to the acceptor brush-border membrane vesicles. Peripheral fluorescence was visible when the brush-border membrane vesicles were viewed with a fluorescent microscope. Using brush-border membrane vesicles from kidneys of vitamin D-deficient animals, quantitation of lipid transfer revealed a 1,25(OH)2D3 (10(-7) M) stimulation of NBD-PC transfer from 1.38 +/- 0.27 to 2.07 +/- 0.26 micrograms/h, and of PC transfer, assessed by vesicle phosphatidylcholine content, from 49.7 +/- 12 to 57.3 +/- 12 micrograms/mg protein per h (P less than 0.05). There was no significant transfer of N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE). In the absence of hormone, the amount of NBD-PC transferred to brush-border membrane vesicles prepared from normal rats was significantly greater than that transferred to brush-border membrane vesicles prepared from vitamin D-deficient animals (2.12 +/- 0.02 vs. 1.39 +/- 0.27 micrograms of NBD-PC/h, P less than 0.05). Both physiologic and pharmacologic concentrations of 1,25(OH)2D3 stimulated NBD-PC transfer with maximum response at 10(-14) M (2.98 +/- 0.15 micrograms/h). 24,25-Dihydroxycholecalciferol and 25-hydroxycholecalciferol (25(OH)D3) also stimulated transfer, although dose-response curves were less effective than for 1,25(OH)2D3. Cortisol and vitamin D-3 did not stimulate transfer. 1,25(OH)2D3 did not stimulate NBD-PC transfer between liposome populations.     

Metabolism of vitamin D3 administered in liposomes in rat liver]

Incorporation of [3H]-cholecalciferol into the liver and the rate of its conversion into 25-OH-D are significantly larger after intravenous administration of this vitamin into liposomes than its dissolution in ethanol. The latter may be due to a different distribution of vitamin D3 in the cells of the liver. Incorporation of [3H]-vitamin D3 in reticulocytes suggests that its cells are a locus for storage of vitamin D, from which the latter is transported into hepatocytes where its 25-hydroxylation proceeds.     

Competitive effect of vitamin D2 and Ca2+ on phospholipid model membranes: an FTIR study

The interaction of Ca(2+), with dipalmitoyl phosphatidylcholine (DPPC) model membranes was studied in the presence and absence of vitamin D(2) by using Fourier transform infrared spectroscopy. Addition of vitamin D(2) and/or Ca(2+) into pure DPPC liposomes shifts the phase transition to higher temperature, orders and decreases the dynamics of the acyl chains in both phases and does not induce hydrogen bond formation in the interfacial region. Moreover, the dynamics of the head group of the phospholipid decreases in both phases. The addition of vitamin D(2) into DPPC liposomes containing Ca(2+), decreases the effect of Ca(2+) at all the functional groups under investigation. Similarly, the effect of vitamin D(2) also decreases in the presence of Ca(2+). This behavior is dominant at high Ca(2+) concentrations. Our results show how simultaneous presence of vitamin D(2) and Ca(2+) alter the behavior of each other, which is reflected as a decrease in the interactions between the ions and vitamin D(2) within the membrane.     

Metabolism of cholesterol, vitamin D3 and 20-hydroxyvitamin D3 incorporated into phospholipid vesicles by human CYP27A1

CYP27A1 is a mitochondrial cytochrome P450 which can hydroxylate vitamin D3 and cholesterol at carbons 25 and 26, respectively. The product of vitamin D3 metabolism, 25-hydroxyvitamin D3, is the precursor to the biologically active hormone, 1α,25-dihydroxyvitamin D3. CYP27A1 is attached to the inner mitochondrial membrane and substrates appear to reach the active site through the membrane phase. We have therefore examined the ability of bacterially expressed and purified CYP27A1 to metabolize substrates incorporated into phospholipid vesicles which resemble the inner mitochondrial membrane. We also examined the ability of CYP27A1 to metabolize 20-hydroxyvitamin D3 (20(OH)D3), a novel non-calcemic form of vitamin D derived from CYP11A1 action on vitamin D3 which has anti-proliferative activity on keratinocytes, leukemic and myeloid cells. CYP27A1 displayed high catalytic activity towards cholesterol with a turnover number (k(cat)) of 9.8 min(-1) and K(m) of 0.49 mol/mol phospholipid (510 μM phospholipid). The K(m) value of vitamin D3 was similar for that of cholesterol, but the k(cat) was 4.5-fold lower. 20(OH)D3 was metabolized by CYP27A1 to two major products with a k(cat)/K(m) that was 2.5-fold higher than that for vitamin D3, suggesting that 20(OH)D3 could effectively compete with vitamin D3 for catalysis. NMR and mass spectrometric analyses revealed that the two major products were 20,25-dihydroxyvitamin D3 and 20,26-dihydroxyvitamin D3, in almost equal proportions. Thus, the presence of the 20-hydroxyl group on the vitamin D3 side chain enables it to be metabolized more efficiently than vitamin D3, with carbon 26 in addition to carbon 25 becoming a major site of hydroxylation. Our study reports the highest k(cat) for the 25-hydroxylation of vitamin D3 by any human cytochrome P450 suggesting that CYP27A1 might be an important contributor to the synthesis of 25-hydroxyvitamin D3, particularly in tissues where it is highly expressed.     

Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin

Vitamin D3 can be hydroxylated sequentially by cytochrome P450scc (CYP11A1) producing 20-hydroxyvitamin D3, 20,23-dihydroxyvitamin D3 and 17,20,23-trihydroxyvitamin D3. The aim of this study was to characterize the ability of vitamin D3 to associate with phospholipid vesicles and to determine the kinetics of metabolism of vitamin D3 by P450scc in vesicles and in 2-hydroxypropyl-beta-cyclodextrin (cyclodextrin). Gel filtration of phospholipid vesicles showed that the vitamin D3 remained quantitatively associated with the phospholipid membrane. Vitamin D3 exchanged between vesicles at a rate 3.8-fold higher than for cholesterol exchange and was stimulated by N-62 StAR protein. The Km of P450scc for vitamin D3 in vesicles was 3.3 mol vitamin D3/mol phospholipid and the rate of conversion of vitamin D3 to 20-hydroxyvitamin D3 was first order with respect to the vitamin D3 concentration for the range of concentrations of vitamin D3 that could be incorporated into the vesicle membrane. 20-Hydroxyvitamin D3 was further hydroxylated by P450scc in vesicles, producing primarily 20,23-dihydroxyvitamin D3, with Km and kcat values 22- and 6-fold lower than those for vitamin D3, respectively. 20,23-dihydroxyvitamin D3 was converted to 17,20,23-trihydroxyvitamin D3 with even lower Km and kcat values. Vitamin D3 and cholesterol were metabolized with comparable efficiencies in cyclodextrin, but the Km for both showed a strong dependence on the cyclodextrin concentration, decreasing with decreasing cyclodextrin. This study shows that vitamin D3 quantitatively associates with phospholipid vesicles, can exchange between membranes, and can be hydroxylated by membrane-associated P450scc but with lower efficiency than for cholesterol hydroxylation. The kcat values for metabolism of vitamin D3 in vesicles and 0.45% cyclodextrin are similar, but the ability to solubilize vitamin D3 at a concentration higher than its Km makes the cyclodextrin system more efficient for producing the hydroxyvitamin D3 metabolites for further characterization.     

Reconstitution in liposomes of the electron-transport chain catalyzing fumarate reduction by formate

Fumarate reduction by formate in Vibrio succinogenes is catalyzed by a membrane-bound electron-transport chain, and is coupled with the phosphorylation of ADP. The electron-transport chain was reconstituted in liposomes from the isolated components. The formate dehydrogenase complex (three different peptides), the fumarate reductase complex (three different peptides) and vitamin K-1 were required for the electron transport. The pathway of the electrons from formate to fumarate in the reconstituted chain was identical with that in the bacterial membrane. Each of the active enzyme complexes in the liposomes participated in the electron transport. This was valid for proteoliposomes with ratios of the contents of the two enzyme complexes ranging between 0.1 and 10. This indicates that vitamin K-1 forms a diffusible pool within the liposomal membrane that allows every quinone molecule to react with each molecule of the two enzyme complexes.     

Membrane permeability changes with vitamin A/vitamin E mixed bilayers

Vitamin A (all trans-retinol) enhances the permeability of egg phosphatidylcholine liposomes to glucose, urea, and erythritol while vitamin E (α-tocopherol) decreases permeability to the same solutes. Egg phosphatidylcholine bilayers containing both vitamin A and vitamin E are shown to have an altered permeability more similar to that affected by vitamin E alone. The membrane stabilizing effect of vitamin E appears dominant over the membrane destabilizing effect of vitamin A.     

Activation of human complement by liposomes: a model for membrane activation of the alternative pathway.

Liposomal model membranes were found to activate the alternative pathway of human complement. Activation was measured by C3 conversion and component consumption in serum that had been incubated with liposomes. C3 conversion did not require C1 or C2 of the classical pathway, since it was observed in serum from a C1r-deficient patient, serum from a C2-dificient patient, and normal serum in buffer containing EGTA and MgCl2. The incubation of liposomes with C2-deficient serum resulted in consumption of components C3 through C9 with no consumption of C1 or C4 in a profile typical of alternative pathwya activation. The reaction was further shown to require alternative pathway factor D, and to be independent of antibody. Activation of the alterative pathway was dependent on the membrane composition of the liposomes. A positive charge was required for liposomes to produce C3 conversion. Liposomal cholesterol concentration and phospholipid fatty acyl chain length and unsaturation all influenced activation, suggesting the importance of membrane fluidity. Positively charged liposomes containing dimyristoyl phosphatidylcholine and cholesterol required the presence of certain glycolipids for C3 conversion. The activation of the alternative complement pathway by liposomes of defined membrane composition may provide a suitable model for the study of alternative pathway activation by cellular membranes.     

Photoconversion of 7-dehydrocholesterol to vitamin D3 in synthetic phospholipid bilayers

The incorporation of 7-dehydrocholesterol into synthetic phospholipid bilayers altered the distribution of products after photolysis. In liposomes, the relative amounts of 7-dehydrocholesterol and lumisterol were elevated, and tachysterol was reduced from the levels observed in hexane solution. Z to E isomerization of the previtamin to tachysterol is favored in organic solvents. The inhibition of this process is evidence that an ordered lipid matrix places a new constraint on the conformation of the ring B fission product--one in which the configuration is favorable for a return to a cyclized diene. Further, rate enhancements of up to 15-fold were observed for the thermal isomerization of the previtamin to vitamin D3 in liposomes. The free energies of activation for the reaction at 25 degrees C were reduced by 1.3-1.5 kcal/mol in the bilayer environment compared to that of hexane. As this reaction involves the concerted transfer of a hydrogen via a cyclic intermediate, it provides additional evidence for membrane stabilization of an all-cis conformation of the previtamin. Photoproduct ratios were also studied for 7-dehydrocholesterol adsorbed to a variety of solid supports. That nonspecific interactions of 7-dehydrocholesterol with lipid can influence product formation may have important implications with respect to the mechanism of vitamin D3 biosynthesis.     

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.     

An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3

Steroid hormones may enter cells by diffusion through the plasma membrane. However, we demonstrate here that some steroid hormones are taken up by receptor-mediated endocytosis of steroid-carrier complexes. We show that 25-(OH) vitamin D3 in complex with its plasma carrier, the vitamin D-binding protein, is filtered through the glomerulus and reabsorbed in the proximal tubules by the endocytic receptor megalin. Endocytosis is required to preserve 25-(OH) vitamin D3 and to deliver to the cells the precursor for generation of 1,25-(OH)2 vitamin D3, a regulator of the calcium metabolism. Megalin-/- mice are unable to retrieve the steroid from the glomerular filtrate and develop vitamin D deficiency and bone disease.     

Effect of phytyl side chain of vitamin E on its antioxidant activity

Inhibition of the oxidation of methyl linoleate and soybean phosphatidylcholine in homogeneous solution and in aqueous dispersion by four chain-breaking antioxidants, vitamin E (alpha-tocopherol), 2,2,5,7,8-pentamethyl-6-chromanol, 2,6-di-tert-butyl-4-methylphenol, and stearyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate, was studied to examine the effect of the phytyl side chain of vitamin E on its antioxidant activity. These four antioxidants exerted similar antioxidative activities. They were also effective as antioxidants in protecting the oxidation of soybean phosphatidylcholine liposomes in water dispersion. However, when they were incorporated into dimyristoyl phosphatidylcholine liposomes, only 2,2,5,7,8-pentamethyl-6-chromanol and 2,6-di-tert-butyl-4-methylphenol could suppress the oxidation of soybean phosphatidylcholine liposomes dispersed in the same aqueous system. It was concluded that the antioxidative properties of vitamin E and its model without the phytyl side chain are quite similar within micelles and liposomes as well as in homogeneous solution but that the phytyl side chain enhances the retainment of vitamin E in liposomes and suppresses the transfer of vitamin E between liposomal membranes.     

Uptake of the hormone 1,25-dihydroxyvitamin D3 by the dog liver

The hepatic uptake of the hormone 1,25-dihydroxyvitamin D3 has been studied, in vivo, using the multiple indicator dilution technique. The fractional uptake of 1,25-dihydroxyvitamin D3 during a single circulatory passage across the dog liver has been estimated at 34.4 +/- 3.3% while its hepatic clearance was estimated at 364.3 +/- 94.1 mL/min. The hepatic uptake of 1,25-dihydroxyvitamin D3 is discussed in relation to its systemic bioavailability following intravenous or oral administration as well as in relation to the hepatic uptake of other vitamin D sterols; it is postulated that the hepatic uptake of vitamin D sterols does not seem to be mediated by specific receptors on the liver plasma membrane; it seems, however, that the hepatic uptake of vitamin D sterols may be inversely related to their relative affinity for the circulating carrier, the vitamin D binding protein.     

Subcellular localization of vitamin D3 25-hydroxylase in human liver

Vitamin D3 25-hydroxylase activity was measured in subcellular and submitochondrial fractions of human liver. Quantitation of 25-hydroxyvitamin D3 was based on high performance liquid chromatography. Vitamin D3 25-hydroxylase activity was detected in the mitochondrial fraction only. The mitochondrial 25-hydroxylase activity was linear with time up to 60 min and with mitochondrial protein up to 1 mg/ml. An apparent Km value of about 10(-5) M was found. Substrate satuation level was not reached. In the presence of 2.4 X 10(-4) M vitamin D3, the rate of 25-hydroxyvitamin D3 formation was 0.19 nmol X mg of protein-1 X h-1 After fractionation of the mitochondria, 86% of the 25-hydroxylase activity was recovered in the mitoplast fraction. The outer membrane fraction was devoid of activity. It is concluded that human liver contains only one detectable vitamin D3 25-hydroxylase enzyme localized to the mitochondrial inner membrane.     

Radical-induced inactivation of kidney Na+,K(+)-ATPase: sensitivity to membrane lipid peroxidation and the protective effect of vitamin E

The Na+,K(+)-ATPase is a membrane-bound, sulfhydryl-containing protein whose activity is critical to maintenance of cell viability. The susceptibility of the enzyme to radical-induced membrane lipid peroxidation was determined following incorporation of a purified Na+,K(+)-ATPase into soybean phosphatidylcholine liposomes. Treatment of liposomes with Fenton's reagent (Fe2+/H2O2) resulted in malondialdehyde formation and total loss of Na+,K(+)-ATPase activity. At 150 microM Fe2+/75 microM H2O2, vitamin E (5 mol%) totally prevented lipid peroxidation but not the loss of enzyme activity. Lipid peroxidation initiated by 25 microM Fe2+/12.5 microM H2O2 led to a loss of Na+,K(+)-ATPase activity, however, vitamin E (1.2 mol%) prevented both malondialdehyde formation and loss of enzyme activity. In the absence of liposomes, there was complete loss of Na+,K(+)-ATPase activity in the presence of 150 microM Fe2+/75 microM H2O2, but little effect by 25 microM Fe2+/12.5 microM H2O2. The activity of the enzyme was also highly sensitive to radicals generated by the reaction of Fe2+ with cumene hydroperoxide, t-butylhydroperoxide, and linoleic acid hydroperoxide. Lipid peroxidation initiated by 150 microM Fe2+/150 microM Fe3+, an oxidant which may be generated by the Fenton's reaction, inactivated the enzyme. In this system, inhibition of malondialdehyde formation by vitamin E prevented loss of Na+,K(+)-ATPase activity. These data demonstrate the susceptibility of the Na+,K(+)-ATPase to radicals produced during lipid peroxidation and indicate that the ability of vitamin E to prevent loss of enzyme activity is highly dependent upon both the nature and the concentration of the initiating and propagating radical species.     

Antioxidant Activity of Vitamin E and Trolox: Understanding of the Factors that Govern Lipid Peroxidation Studies In Vitro

Peroxidation of lipids is of significant interest owing to the evidence that peroxyl radicals and products of lipid peroxidation may be involved in the toxicity of compounds initiating a deteriorative reaction in the processing and storage of lipid-containing foods. In view of the significance of the antioxidant role of the dietary compound vitamin E and its water-soluble analogue Trolox in research of lipid-containing foods, it is desirable to determine more specifically how and where they operate its antioxidant activity in lipid membranes. In this study, unilamellar liposomes of phosphatidylcholine were used as membrane mimetic systems to estimate the antioxidant properties of vitamin E and Trolox and establish a relationship between their interactions with the membrane and their consequent antioxidant activity. Lipid peroxidation was initiated by the peroxyl radical (ROO^•) in lipid and aqueous media by the thermal decomposition of azocompounds and was assessed by the fluorescence intensity decay of the fluorescent probe diphenylhexatriene propionic acid. Results obtained showed that membrane lipoperoxidation is related not only to the scavenging characteristics of the compounds studied but also to their ability to interact with the lipid bilayers, and consequently liposomes provide additional information to that obtained currently from assays performed in aqueous buffer media.     

Annexin II is the membrane receptor that mediates the rapid actions of 1alpha,25-dihydroxyvitamin D(3)

1alpha,25-Dihydroxyvitamin D(3) has been shown to exert its effects by both genomic (minutes to hours) and rapid (seconds to minutes) mechanisms. The genomic effects are mediated by interaction with the nuclear vitamin D receptor. We show that the vitamin D analog, [(14)C]-1alpha,25-dihydroxyvitamin D(3) bromoacetate, is specifically bound to a protein (molecular weight 36 kDa) in the plasma membrane of rat osteoblastlike cells (ROS 24/1). The plasma membrane protein labeled with the bromoacetate analog was identified as annexin II by sequence determination and Western blot. Partially purified plasma membrane proteins (PI 6.9-7.4) and purified annexin II exhibited specific and saturable binding for [(3)H]-1alpha, 25-dihydroxyvitamin D(3). Antibodies to annexin II inhibited [(14)C]-1alpha,25-dihydroxyvitamin D(3) bromoacetate binding to ROS 24/1 plasma membranes, immunoprecipitated the ligand-protein complex, and inhibited 1alpha,25-dihydroxyvitamin D(3)-induced increases in intracellular calcium in ROS 24/1 cells. The results indicate that annexin II may serve as a receptor for rapid actions of 1alpha, 25-dihydroxyvitamin D(3).     

Vitamin D3 administration increases the membrane fluidity of intestinal mitochondria

The fluorescence anisotropy in the mitochondria from vitamin D-treated chicks is significantly lower than that from the vitamin D-deficient animals with the inner core probe DPH. Surface membrane fluidity, measured with the probe TMA-DPH, shows no differences between the organelles of both groups. The fluorescence studies performed in mitochondrial subfractions revealed that cholecalciferol treatment induces a decrease of lipid order parameter S (DPH) in the mitochondrial inner membrane. These results pose the question of whether vitamin D3 participates in the regulation of physiological function of the intestinal mitochondria through changes in the physical properties of the membranes.     

Liver microsomal membrane fluidity and lipid characteristics in vitamin A-deficient rats.

Rat liver microsomes (microsomal fraction) were isolated from vitamin A-deficient and -sufficient rats and analysed for membrane lipid characteristics. Membrane fluidity was found to be significantly decreased in microsomes from the vitamin A-deficient rats, but not in liposomes prepared from lipid extracts. Microsomes from vitamin A-deficient animals showed a significant decrease in C18:2, omega 6 and an increase in C22:5, omega 6 fatty acids.