A Liposomal Model That Mimics the Cutaneous Production of Vitamin D3
STUDIES OF THE MECHANISM OF THE MEMBRANE-ENHANCED THERMAL ISOMERIZATION OF PREVITAMIN D3 TO VITAMIN D3*

Xiao Quan TianDagger and Michael F. Holick§

From the Vitamin D, Skin, and Bone Research Laboratory, Endocrinology Nutrition and Diabetes Section, § Departments of Medicine and Dagger  Physiology, Boston University Medical Center, Boston, Massachusetts 02118

    ABSTRACT
Top
Abstract
Introduction
References

We reported previously that the rate of previtamin D3 (preD3) right-left-harpoons  vitamin D3 isomerization was enhanced by about 10 times in the skin compared with that in organic solvents. To elucidate the mechanism by which the rate of this reaction is enhanced in the skin, we developed a liposomal model that mimicked the enhanced isomerization of preD3 to vitamin D3 that was described in human skin. Using this model we studied the effect of changing the polarity of preD3 as well as changing the chain length and the degree of saturation of liposomal phospholipids on the kinetics of preD3 right-left-harpoons  vitamin D3 isomerization. We found that a decrease in the hydrophilic interaction of the preD3 with liposomal phospholipids by an esterification of the 3beta -hydroxy of preD3 (previtamin D3-3beta -acetate) reduced the rate of the isomerization by 67%. The addition of a hydroxyl on C-25 of the hydrophobic side chain (25-hydroxyprevitamin D3), which decreased the hydrophobic interaction of preD3 with the phospholipids, reduced the rate by 87%. In contrast, in an isotropic n-hexane solution, there was little difference among the rates of the conversion of preD3, its 3beta -acetate, and 25-hydroxy derivatives to their corresponding vitamin D3 compounds. We also determined rate constants (k) of preD3 right-left-harpoons  vitamin D3 isomerization in liposomes containing phosphatidylcholines with different carbon chain lengths. The rates of the reaction were found to be enhanced as the number of carbons (Cn) in the hydrocarbon chain of the phospholipids increased from 10 to 18. In conclusion, these results support our hypothesis that amphipathic interactions between preD3 and membrane phospholipids stabilize preD3 in its "cholesterol like" cZc-conformer, the only conformer of preD3 that can convert to vitamin D3. The stronger these interactions were, the more preD3 was likely in its cZc conformation at any moment and the faster was the rate of its conversion to vitamin D3.

    INTRODUCTION
Top
Abstract
Introduction
References

During evolution, many poikilothermic and homeothermic terrestrial vertebrates including humans acquired the ability to photosynthesize vitamin D3 in their skin (1, 2) and to use vitamin D3 to enhance the efficiency of dietary calcium absorption to maintain a healthy and mineralized skeleton (3, 4). Cutaneous synthesis of vitamin D3 consists of both photo- and thermal reactions (5, 6) (Fig. 1). When exposed to sunlight, ultraviolet-B (UV-B) (290-315 nm) radiation photolyzes provitamin D3 (7-dehydrocholesterol (7-DHC)1) (Fig. 1A), a Delta 5,7-sterol synthesized in the skin, into a 9,10-seco B sterol, cZc-previtamin D3 (cZc-preD3) (Fig. 1B). This novel structure enables the seco-sterol to change its configuration between the preD form and the vitamin D form via a 1,7-sigmatropic hydrogen shift (Fig. 1, B and C) (7-11), one of the pericyclic processes defined by Woodward and Hoffmann (8). This thermally dependent reaction is the final step to produce vitamin D3 in the skin and represents one of the best known examples of a concerted reaction that occurs in vivo. Although it has long been noted that, like other concerted reactions, the interconversion between preD3 right-left-harpoons  vitamin D3 is not influenced by a solvent effect when carried out in isotropic solutions (12), our recent data have revealed that both kinetics and thermodynamics of this reaction may change significantly in many anisotropic microenvironments (13, 14). Therefore, this simple yet physiologically important reaction provides us an ideal model to study the mechanism by which the kinetics of a concerted reaction is modified by an anisotropic medium. The seco-B ring of preD3 consists of a conjugated triene system, which confers preD3 with high conformational mobility (Fig. 1). Although the middle double bond in the triene system of preD3 is in the cis (Z) configuration, two conformations arise from rotation around the single bonds C5-C6 and C7-C8 within the triene system i.e. cZc or s-cis,s-cis conformations (Fig. 1B) and tZc or s-trans,s-cis conformations (Fig. 1D) (9-11). It is known that to chemically isomerize to vitamin D3, preD3 is required to be in the cZc conformation (Fig. 1B) (9-11). From a structure-reactivity point of view, it is important to know whether there is any effect of the conformational restraints imposed by anisotropic media on the chemical transformation of preD3 into vitamin D3. We have previously shown that the rate of the formation of vitamin D3 from preD3 was enhanced by about 10-fold in the skin of terrestrial vertebrates compared with the rate of the same isomerization in an isotropic solution (1, 2, 13). Based on the finding that the major fraction of cellular 7-DHC and preD3 are present in the cell membrane (13), we proposed a mechanism for the membrane-enhanced isomerization of preD3 to vitamin D3. We hypothesized that within the anisotropic membrane bilayers, amphipathic interactions between preD3 and phospholipids stabilized the "cholesterol-like" cZc conformation of preD3 (Fig. 2) and shifted the conformational equilibrium of preD3 toward cZc-preD3, the only conformation of preD3 that isomerizes to vitamin D3. Therefore, the rate of preD3 right-left-harpoons  vitamin D3 reaction is enhanced. To test this hypothesis, the effect of changing the polarity of 7-DHC as well as changing the chain length and the degree of saturation of the liposomal phospholipids on the kinetics of preD3 right-left-harpoons  vitamin D3 reaction were investigated. We have demonstrated for the first time that there is a positive correlation between the strength of the amphipathic interactions of preD3 with liposomal phospholipids and the rate of preD3 right-left-harpoons  vitamin D3 isomerization.


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Fig. 1.   Photolysis of 7-dehydrocholesterol and its analogs to previtamin D3 and its analogs and their subsequent thermal isomerization to the corresponding vitamin D3 forms. A, R = H; X = H: 7-dehydrocholesterol; R = H, X = O: 25-hydroxy-7-dehydrocholesterol; R = CH3CO-, X = H: 7-dehydrocholesterol-3beta -acetate. B, R = H, X = H: cZc-previtamin D3; R = H, X = OH: cZc-25-hydroxyprevitamin D3; R = CH3CO-, X = H: cZc-previtamin D3-3beta -acetate. C, R = H, X = H: vitamin D3; R = H, X = OH: 25-hydroxyvitamin D3; R = CH3CO-; X = H: vitamin D3-3beta -acetate; D, R = H; X = H: tZc-previtamin D3; R = H, X = OH: tZc-25-hydroxyprevitamin D3; R = CH3CO-, X = H: tZc-previtamin D3-3beta -acetate.


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Fig. 2.   Proposed theoretical structural model for the localization of the cZc-previtamin D3 in the phospholipids of a membrane. Based on the amphipathic nature and conformational mobility of previtamin D3, we proposed the following model to show the spatial relationship between previtamin D3 and phospholipids. We postulated that in the membrane, the cholesterol like cZc-previtamin D3 is aligned parallel to its neighboring phospholipids, with its polar 3beta -hydroxy interacting with the polar head groups of the phospholipids through hydrogen bonding, and the hydrophobic rings and side chain interacting with the nonpolar acyl chains of the lipids through hydrophobic and van der Waals interactions. A, phosphatidylcholine; B, cZc-previtamin D3.


    EXPERIMENTAL PROCEDURES

Materials

7-DHC and vitamin D3 were purchased from Sigma. 7-DHC-3beta -acetate was purchased from Steroids Inc. (Wilton, NH). 25-Hydroxy-7-dehydrocholesterol (25(OH)7-DHC) was a kind gift from Dr. Rick Gray (Amoco Bioproducts, Naperville, IL). All the above chemicals were purified by high performance liquid chromatography (HPLC) and stored at -80 °C before use. Saturated lipids including didecanoylphosphatidylcholine (DDPC, C10:0) (99%), dilauroylphosphatidylcholine (DLPC, C12:0) (99%), dipalmitoylphosphatidylcholine (DPPC, C16:0) (99%), distearoylphosphatidylcholine (DSPC, C18:0) (99%), diarachidoylphosphatidylcholine (DAPC, C20:0) (99%), and unsaturated lipids dipalmitoleoylphosphatidylcholine (C16:1, [cis]-9) (99%) were obtained from Sigma.

HPLC was performed with a P1000 pump equipped with a UV2000 UV-visible absorption detector (Thermo Separation Products, San Jose, CA). An Econosphere silica column (250 × 4.6 mm, 5 µm; Alltech Associate, Inc., Deerfield, IL) was used to separate the various vitamin D metabolites except their 3beta -acetate derivatives, which were separated by a Cyclobond I 2000 column (100 × 4.6 mm, 5 µm; Advanced Separation Technologies Inc., Whippany, NJ).

UV spectra of vitamin D3, preD3 and their photoisomers as well as their 25-hydroxylated derivatives were recorded either by a UV spectrometer (U-2000, Hitachi Instruments, Inc., Stoughton, MA) or by an online HPLC UV detector (UV2000).

Methods

Preparation of Liposomes and Human Skin Samples-- Liposomes were prepared by a modified procedure reported by Wiseman et al. (15, 16), whereby 7-DHC and vitamin D3 were incorporated into liposomes. The different lipids were dissolved in pure chloroform to give a final concentration of 20 mg/ml. An aliquot of the stock solution was taken and mixed with an equal volume of one of the following standard solutions of 7-DHC, 25(OH)7-DHC, and 7-DHC-3beta -acetate. The organic solvent was evaporated by a stream of nitrogen. When necessary, the vials containing lipids were left under high vacuum overnight to remove trace solvent. The thin lipid film was resuspended in a 10 mM phosphate buffer (pH = 7.4) followed by vortexing and sonication. The resulting liposomes (lipid concentration, 6.75 mM; molar ratio of lipid to incorporated sterol, 100:2) were sealed in ampules that had previously been flushed with argon. The preparation of liposomes (hydration and vortexing) was performed above the transition temperature (Tm) of the corresponding lipid mixture (DMPC, 23 °C; DPPC, 41.5 °C; and DSPC, 58 °C) (17).

Human skin samples were prepared by a previously described method (5, 13). Briefly, neonatal foreskin was cleaned from subcutaneous tissues and cut into small pieces (about 0.3 cm2). Before UV irradiation, skin samples were immersed in a water bath at 60 °C for 30 s (this technique allows the separation of the dermis from epidermis at the stratum basale).

Photoreaction and Thermal Isomerization-- Heat-treated skin samples, ampules containing liposome preparations, ampules containing a solution of 7-DHC or its analogues in n-hexane (0.135 mM), and ampules containing a solution of 7-DHC-DPPC in n-hexane (7-DHC, 0.135 mM; DPPC, 6.75 mM) were placed on ice and irradiated by UV Medical Sunlamps (National Biological Corp., Cleveland, OH) for 3 min (13). Spectral output for these lamps peaked at about 314 nm, and about 56% of the total output was within UV-B range (13). UV-irradiated samples were incubated at 37 °C, and aliquots were taken at different time intervals. The formation of vitamin D3 from preD3 was analyzed by the HPLC method described below.

HPLC Analysis-- Immediately after incubation, the heat-treated skin was separated into epidermis and dermis according to a previously described method (5). Epidermis and liposomes were extracted three times with 8% ethyl acetate in n-hexane (5, 13). The organic phase (upper layer) was transferred into a test tube and dried under a stream of nitrogen. The residue was reconstituted with mobile phase and centrifuged briefly to remove insoluble particles before HPLC analysis.

Methods of HPLC separation and quantification of unmodified preD3 and vitamin D3 have been described previously (2, 13, 14). To the best of our knowledge, there have been no reports on HPLC separation of 25(OH)preD3 as well as preD3 acetate from their photo- and thermal isomers in the literature. We therefore developed the following HPLC systems for the separations. Complete separation of 25(OH)preD3 from its photo- and thermal isomers was obtained with isocratic elution of 2% 2-propanol in n-hexane on an Alltech Econosphere silica column (250 × 4.6 mm, 5 µm). Separation of the various preD3 acetate isomers was achieved by using an Astec Cyclobond I 2000 column (100 × 4.6 mm, 5 µm) with 0.03% 2-propanol in n-hexane as a mobile phase.

Kinetic Studies of the Thermal Isomerization-- The thermal conversion between preD3 and vitamin D3 is a first order reversible process. The integrated rate equation is expressed as
k=1/t <UP>ln</UP>[(<UP>D</UP><SUB>e</SUB>−<UP>D</UP><SUB>o</SUB>)/(<UP>D</UP><SUB>e</SUB>−<UP>D</UP><SUB>t</SUB>)] (Eq. 1)
where De, Do, and Dt are concentrations of vitamin D3 at time t equals equilibrium, zero and t, respectively. The total rate constant (k) is calculated from the slope of the plot of ln[(De - Do)/(De - Dt)] versus time t. The equilibrium constant (K) of the reaction is expressed as:
K=[<UP>vitamin</UP> D]<SUB><UP>eq</UP></SUB>/[<UP>preD</UP>]<SUB><UP>eq</UP></SUB> (Eq. 2)
where [vitaminD]eq and [preD]eq are equilibrium concentrations of vitamin D3 and preD3, respectively.

    RESULTS

Separation of Vitamin D3, Previtamin D3, and Its Photoisomers as Well as Their 25-Hydroxylated Derivatives-- PreD3 was completely separated from its photoisomers and vitamin D3, as described previously (13). 25-Hydroxyprevitamin D3 (25(OH)preD3), 25-hydroxyvitamin D3 (25(OH)D3), 25(OH)7-DHC, 25-hydroxylumisterol3 (25(OH)L3), and 25-hydroxytachysterol3 (25(OH)T3) were completely separated from each other by HPLC (Fig. 3). Peak identities of 25(OH)7-DHC and 25(OH)D3 were assigned on the coelution of both compounds with standards on both normal and reversed phase HPLC. 25(OH)L3 and 25(OH)T3 were identified by their characteristic UV absorption spectra (3, 5, 6). 25(OH)preD3 was identified by both its UV absorption spectrum (lambda max = 260 nm) and its ability to thermally isomerize to 25(OH)D3. Base-line separation of vitamin D3-3beta -acetate, preD3-3beta -acetate, and 7-DHC-3beta -acetate was accomplished with the normal phase HPLC method described under "Experimental Procedures."


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Fig. 3.   HPLC separation of 25-hydroxy-7-dehydrocholesterol, its photoisomers, and 25-hydroxyvitamin D3. Peak a, 25(OH)L3; peak b, 25(OH)D3; peak c, 25(OH)preD3; peak d, 25(OH)7-DHC, and peak e, 25(OH)T3.

Kinetics of PreD3 right-left-harpoons  Vitamin D3 Reaction in Isotropic (n-Hexane and DPPC/n-Hexane) Versus in Anisotropic Microenvironments (Human Skin and DPPC Liposomes)-- Solutions of 7-DHC in n-hexane (0.135 mM), 7-DHC in DPPC/n-hexane (7-DHC, 0.135 mM; DPPC, 6.75 mM), DPPC liposomes containing 7-DHC (7-DHC, 0.135 mM; DPPC, 6.75 mM, i.e. 2 mol % of 7-DHC) as well as samples of human skin were exposed to UV radiation on ice to generate preD3. The isomerization of preD3 into vitamin D3 was followed by means of HPLC analysis. The results showed that the reaction was of first order whether it was carried out in n-hexane, DPPC/n-hexane, skin, or liposomes (Fig. 4). The rate constants of the isomerization were calculated from the integrated rate equation (Eq. 1) using the least squares method. It was found that in anisotropic microenvironments (human skin and DPPC liposomes), the rates of the isomerization were about 10 times larger than those in isotropic ones (n-hexane and DPPC/n-hexane). At 37 °C, they were 8.62 ± 0.24 × 10-5 s-1 (regression coefficient, r = 0.999) and 8.72 ± 1.11× 10-5 s-1 (r = 0.990) in human skin and DPPC liposomes, respectively, versus 8.08 ± 0.07 × 10-6 s-1 (r = 0.999) and 8.06 ± 0.13 × 10-6 s-1 (r = 0.995) in n-hexane and DPPC/n-hexane, respectively. The kinetic plot of the isomerization is shown in Fig. 4. The equilibrium constants (defined by the ratio k1/k2) were calculated from Eq. 2. They were 11 and 10 in human skin and DPPC liposomes, respectively, versus 6 and 7 in n-hexane and DPPC/n-hexane, respectively.


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Fig. 4.   Kinetic plot of preD3 right-left-harpoons  vitamin D3 isomerization in isotropic (n-hexane and DPPC/n-hexane solution) versus anisotropic microenvironments (human skin and DPPC liposomes).

Temperature Dependence on Rate Constants of the Isomerization in DPPC Liposomes-- The rate constants of preD3 right-left-harpoons  vitamin D3 reaction in DPPC liposomes was determined at 0, 15, 30, 37, 50, 60, and 70 °C, and they were 2.40 × 10-6 s-1, 1.25 × 10-5 s-1, 4.26 × 10-5 s-1, 8.72 × 10-5 s-1, 2.12 × 10-4 s-1, 4.37 × 10-4 s-1, 1.00 × 10-3 s-1, respectively. The activation energy for the isomerization was determined according to the Arrhenius equation,
<UP>ln</UP> k=<UP>ln</UP> A−E<SUB>a</SUB>/<UP>R</UP>T (Eq. 3)
where k is the rate constant for the isomerization, ln A is a constant, Ea is activation energy, R is the molar gas constant, and T is the temperature in degrees Kelvin. The Arrhenius plot (Fig. 5) for the isomerization in DPPC liposomes showed a straight line (r = -0.999) for the entire temperature range examined, which included temperatures well above or below the phase transition temperature (Tm) of DPPC liposomes (Tm = 41.5 °C). This result indicated that the mechanism for the isomerization remained the same whether the DPPC liposomes were in the liquid-crystalline phase or in the gel phase. The calculated Ea for the isomerization in DPPC liposomes is 66 kJmol-1, which is similar to the value determined in human skin (73 kJmol-1), and both of them are significantly lower than that determined in n-hexane (87 kJmol-1) (13).


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Fig. 5.   Arrhenius plot of preD3 right-left-harpoons  vitamin D3 isomerization in DPPC liposomes.

Kinetics of the Isomerization of PreD3 and Its Analogues in n-Hexane and DPPC Liposomes-- Ampules containing 7-DHC, 25(OH)7-DHC, and 7-DHC-3beta -acetate in n-hexane and in DPPC liposomes were placed on ice and irradiated with UV radiation to generate preD3 and preD3 analogues. Immediately after irradiation, the exposed ampules were incubated at 37 °C. The formation of vitamin D3 from preD3 was monitored by HPLC. It was found that in an isotropic n-hexane solution, a decrease in the polarity of preD3 at the C3beta position by esterification (preD3-3beta -acetate) or an increase in polarity with the addition of a hydroxyl at C25 (25(OH)preD3) had little effect on the rate of the thermal isomerization. The rates for the isomerization of preD3, preD3-3beta -acetate, and 25(OH)preD3 in an isotropic solution (n-hexane) were 8.08 ± 0.07 × 10-6 s-1, 7.05 ± 0.42 × 10-6 s-1, and 8.36 ± 0.07 × 10-6 s-1, respectively (Fig. 6A). The rates of the thermal isomerization of these preD3 derivatives were drastically different from each other when incorporated into liposomes (Fig. 6B). In liposomes, the unmodified preD3 had the highest rate of the isomerization, k = 8.72 ± 1.11 × 10-5 s-1. In contrast, the rates for the isomerization of preD3-3beta -acetate and 25(OH)preD3 in liposomes were greatly reduced by 67% (k = 2.86 ± 0.40 × 10-5 s-1) and 87% (1.16 ± 0.30 × 10-5 s-1), respectively.


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Fig. 6.   Kinetic plot of the isomerization of preD3 (bullet ), 25(OH)preD3 (triangle ), and preD3-3beta -acetate () to their corresponding vitamin D3 analogs in n-hexane (A) and in DPPC liposomes (B).

Effects of Lipid Composition of Liposomes on the Rate of the Isomerization-- To examine the effects of lipid composition of liposomes on the rate of preD3 right-left-harpoons  vitamin D3 conversion, 7-DHC was incorporated into liposomes containing phospholipids varying in hydrocarbon chain length i.e. DDPC (C10:0), DLPC (C12:0), DPPC (C16:0), DSPC (C18:0), and DAPC (C20:0). 7-DHC was also incorporated into the liposomes made of cis-unsaturated phospholipids, i.e. dipalmitoleoylphosphatidylcholine (C16:1, [cis]-9). Ampules containing each of the above liposomal preparations were placed on ice and irradiated with UV radiation to produce preD3. Exposed samples were incubated at 37 °C for different time intervals. The formation of vitamin D3 from preD3 showed that the rate of the isomerization correlated positively with the length of the hydrocarbon chain of lipids from C10 to C18 and followed the order DDPC (C10:0) (k = 3.93 ± 0.05 × 10-5 s-1) < DLPC (C12:0) (k = 5.13 ± 0.16 × 10-5 s-1) < DPPC (C16:0) (k = 8.72 ± 1.11 × 10-5 s-1) <DSPC (C18:0) (k = 1.04 ± 0.02 × 10-4 s-1). The linear regression equation for the rate constants of the isomerization versus carbon chain length of lipids (Fig. 7) was deduced from the above data and was written as
<UP>ln</UP> k=<UP>−</UP>14.02+1.68 <UP>ln C</UP><SUB>n</SUB> (Eq. 4)
where k (s-1) was the rate constant of the isomerization, and Cn was the number of carbons in the hydrocarbon chains of phosphatidylcholines. The correlation coefficient (r) of the determined regression equation was 0.999. Eq. 4 allows the calculation of rate constants of the isomerization in various liposomes and is valid for liposomal lipids containing 10 to 18 carbon atoms in hydrocarbon chain. Increasing the length of hydrocarbon chain further from C18 to C20 did not enhance but decreased the rate constant (kC18 = 1.04 ± 0.02 × 10-4 s-1 versus kC20 = 8.01 ± 0.08 × 10-5 s-1). The effect of incorporating cis-unsaturated phospholipids into the liposomes on the rate of the isomerization was also examined. It was found that by introducing a cis double bond at carbon 9 of hydrocarbon tails of DPPC liposomes, the rate of the reaction was significantly reduced by more than 40%, i.e. k = 4.86 ± 0.63 × 10-5 s-1 for liposomes prepared by unsaturated phospholipids (C16:1, cis-9) versus k = 8.72 ± 1.11 × 10-5 s-1 for liposomes constructed by saturated phospholipids of the same carbon chain length (DPPC, C16:0).


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Fig. 7.   Effects of phospholipid carbon chain length and saturation on the rate of preD3 right-left-harpoons  vitamin D3 isomerization in liposomes. k, rate constant; n, carbon number of phospholipid chain.


    DISCUSSION

The first objective of this study was to develop a simple model by which the mechanism of the synthesis of vitamin D3 in the skin could be studied. Based on our previous finding that 7-DHC and its photolyzed product preD3 were present in the skin cell membrane, we incorporated 7-DHC into liposomes to mimic the membrane phospholipid bilayers. We found that the rate for the preD3 right-left-harpoons  vitamin D3 isomerization in DPPC liposomes and human skin was essentially the same. The rate of the reaction was about 10-fold faster in liposomes (8.72 ± 1.11 × 10-5 s-1) compared with that in n-hexane (8.08 ± 0.07 × 10-6 s-1) and similar to human skin (8.62 ± 0.24 × 10-5 s-1). The equilibrium constant was also significantly higher in liposomes than the values obtained from the isotropic n-hexane solution and similar to human skin. Thus our liposome model closely mimicked the kinetic properties of the preD3 right-left-harpoons  vitamin D3 isomerization in the skin (1, 2, 13). This is in contrast to the cholesteric liquid-crystalline model used by Cassis and Weiss (18) in which the determined equilibrium constant was smaller instead of larger than that determined in organic solvents.

Next, we examined the effects of isotropic versus anisotropic interactions of DPPC with preD3 on the rate of preD3 right-left-harpoons  vitamin D3 isomerization. We found that in an isotropic solution of DPPC/n-hexane, the rate of preD3 right-left-harpoons  vitamin D3 isomerization is essentially the same as in a solution of pure n-hexane (8.06 ± 0.13 × 10-6 s-1 versus 8.08 ± 0.07 × 10-6 s-1) (Fig. 4). In contrast, the rate of preD3 right-left-harpoons  vitamin D3 isomerization in a solution of DPPC liposomes is enhanced by about 10 times compared with that in n-hexane (Fig. 4). Our results indicate that isotropic interactions of phospholipids with preD3 do not affect the rate of preD3 right-left-harpoons  vitamin D3 conversion, but in an anisotropic microenvironment such as liposomes and skin, the rate of the isomerization is greatly enhanced (Fig. 4).

We used the liposomes as a simple model for the membrane bilayers to study the mechanism by which the rate of preD3 right-left-harpoons  vitamin D3 conversion was greatly enhanced in the skin. It is known that phospholipids and preD3 are amphipathic molecules (ones with both hydrophilic and hydrophobic parts). Based on structural similarity, it was proposed (1, 19, 20) that, within the ordered lipid bilayer, preD3 molecules oriented themselves in a way similar to cholesterol molecules, i.e. with their hydrophilic C3beta hydroxyl group close to the polar head groups of lipids, whereas their hydrophobic rings and the side chain aligned along the hydrocarbon chains of membrane lipids (Fig. 2). However, unlike the rigid cholesterol, the seco-B ring sterol, preD3, is very conformationally mobile. Rotation about the single C5-C6 bond of preD3 generates a wide array of conformations extending from cZc-preD3 to tZc-preD3 (Fig. 1). It was well established that the thermal isomerization between preD3 right-left-harpoons  vitamin D3 was a conformation-controlled process and required a cyclic transition state, possible only for cZc conformers but not possible for tZc and other conformers (Fig. 1) (10, 11).

We hypothesized that the conformational restraints imposed by amphipathic interactions between preD3 and phospholipids stabilized the "cholesterol like" cZc-preD3 conformer (Fig. 2). Therefore, the conformational equilibrium of cZc-preD3 right-left-harpoons  tZc-preD3 was shifted to the cZc conformation. According to the hypothesis, the population of preD3 molecules existing in the cZc form at any instant was higher in a lipid bilayer compared with an isotropic solution, and consequently, the rate of the thermal isomerization of preD3 was enhanced (1). If this hypothesis was correct, it was expected that there would be a positive correlation between the strength of the amphipathic interactions and the rate of this thermal isomerization. To evaluate the hypothesis, we examined the effect of decreasing the polarity of the preD3 by acetylation of C3beta -OH on the kinetics of the thermal conversion of preD3 in liposomes. It was found that by reducing the strength of the amphipathic interactions between the amphiphiles, the rate of the isomerization of preD3 was markedly reduced. The rate of the isomerization of preD3-3beta -acetate was reduced to less than one-third the rate of its unesterfied counterpart, preD3 (Fig. 6). The reduced rate of the isomerization probably reflected that the less polar C3beta ester group was less effective than free C3beta -OH of preD3 in hydrophilic interaction with polar head groups of liposomal phospholipids.

We speculated that the free 3beta -OH of preD3 would interact with adjacent polar phospholipid head group via hydrogen bonding as cholesterol likely does (21, 22). Hydrogen bonding can lead to tight packing of phospholipid bilayers and, thus, enhance the strength of the amphipathic interaction of liposomal lipids with preD3. Because of the absence of a free 3beta -OH, preD3-3beta -acetate was unable to form a hydrogen bond with the carbonyl groups of phospholipids. This weakened the hydrophilic interactions between preD3-3beta -acetate, and membrane lipids probably reduced the efficiency of stabilizing the preD3-3beta -acetate in its cZc conformer and led to a slower rate of its isomerization to vitamin D3-3beta -acetate. This observation emphasizes the important role of C3 hydroxyl group for the correct orientation of the preD3 molecule within phospholipid bilayers and its optimal hydrophilic interactions with polar head groups of membrane phospholipids.

We also investigated whether a disruption in the hydrophobic (van der Waals) interactions between preD3 and hydrophobic tails of phospholipids had an effect on the rate of preD3 right-left-harpoons  vitamin D3 conversion by the addition of a hydroxyl group on the hydrophobic side chain of preD3 at C25. It is known that the liposomal phospholipid bilayers are held together mainly by hydrophobic interactions. The presence of hydrophilic C25 hydroxyl group in the hydrophobic core disorganized phospholipid bilayers (22, 23), and thus, the strength of van der Waals interactions between the hydrophobic tails of phospholipids and 25(OH)preD3 could be significantly weakened. It is also possible that 25(OH)preD3 can be folded so that both 3beta - and 25-hydroxyl groups align close to polar head groups of liposomal phospholipids (23) and further disorganize phospholipid bilayers. Accordingly, the stabilizing effect of phospholipid bilayers on the cZc-25(OH)preD3 would be much less effective. Therefore, the rate of the 25(OH)preD3 right-left-harpoons  25(OH)D3 isomerization was anticipated to be reduced more markedly than the rate of preD3-3beta -acetate right-left-harpoons  vitamin D3-3beta -acetate isomerization. Our results demonstrated that the rate of the isomerization of 25(OH)preD3 in liposomes was reduced by about 7-fold compared with the rate of the isomerization of preD3 in liposomes, which approached the rate determined in an isotropic solution (Fig. 6).

The strength of the amphipathic interactions between the incorporated preD3 and liposomal phospholipids depends not only on the polarity of preD3 but also on the hydrophobicity of phospholipids. To evaluate the effect of changing structures of phospholipids by altering either the carbon chain length or chain saturation on the rate of preD3 right-left-harpoons  vitamin D3, isomerization was examined.

We first carried out kinetic studies of preD3 right-left-harpoons  vitamin D3 isomerization in liposomes prepared by phospholipids with different hydrocarbon chain lengths. We observed a chain length-dependent rate enhancement of the isomerization. Fig. 7 showed that the rate of the thermal isomerization in the different liposomes followed the order DSPC (C18:0) > DPPC (C16:0) > DLPC (C12:0) > DDPC (C10:0). Least squares analysis of the data revealed a positive linear relationship (r = 0.999) between ln k (k, rate constant) and ln Cn (Cn, number of carbon atoms in the hydrocarbon chains of phospholipids) (Fig. 7). Because the strength of the amphipathic interactions is directly related to the chain length of phospholipids, our observation further supports the hypothesis that there is a positive correlation between the strength of the anisotropic interactions between the amphiphiles and the rate of the isomerization.

We found that DSPC (C18:0) liposomes had the maximum effect on the rate enhancement of preD3 right-left-harpoons  vitamin D3 isomerization (Fig. 7). It indicated that the optimal amphipathic interactions between preD3 and phospholipids were achieved in DSPC (18:0) liposomes. Based on this information, we estimated that the "effective hydrophobic length of preD3" corresponded to the length of a 18-carbon chain, which was similar to the reported value of cholesterol in phospholipid bilayers (the length of a 17-carbon chain or about 17.5 angstroms) (24-27).

To gain further insight into the mechanism of membrane-enhanced preD3 right-left-harpoons  vitamin D3 isomerization, we carried out kinetic studies of the reaction in liposomes prepared with unsaturated phospholipids. We found that the rate of the isomerization was reduced by more than 40% when the reaction was carried out in C16:1 (cis-9) liposomes compared with the reaction in saturated C16:0 liposomes. It is known that the cis double bonds cause rigid kinks of 30° in the hydrocarbon chains. These kinks cause disorder in the packing of the hydrophobic chains and increase the distance between hydrocarbon chains of phospholipids and the incorporated preD3, which are expected to reduce the strength of van der Waals interaction between these amphiphiles (28). This reduced van der Waals interaction would be less effective in stabilizing the cholesterol-like cZc-preD3 conformer. Accordingly, the rate of the isomerization was more than 40% slower in cis-unsaturated liposomes than that in saturated ones of comparable carbon chain length (Fig. 7).

We concluded that in an ordered lipid bilayer, conformational restraints imposed by amphipathic interactions stabilized the cholesterol-like cZc conformer of preD3. The stronger the amphipathic interactions were, the more preD3 was in cZc conformation, and the faster was the rate of its conversion to vitamin D3.

    ACKNOWLEDGEMENTS

We thank Dr. James A. Hamilton for his critiques and recommendations on experimental design of liposomal studies and Dr. David M. Jackson for his help for preparing the figures.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant RO1-AR-36963 and NASA Grant NAGW4936.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 617-638-4545; Fax: 617-638-8882; E-mail: mfholick{at}bu.edu.

The abbreviations used are: 7-DHC, 7-dehydrocholesterol; preD3, previtamin D3; 25(OH)7-DHC, 25-hydroxy-7-dehydrocholesterol; 25(OH)L3, 25-hydroxylumisterol3; 25(OH)T3, 25-hydroxytachysterol3; HPLC, high performance liquid chromatography; DDPC, didecanoylphosphatidylcholine; DLPC, dilauroylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DSPC, distearoylphosphatidylcholine; DAPC, diarachidoylphosphatidylcholine.
    REFERENCES
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Abstract
Introduction
References

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