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
Tian
and
Michael F.
Holick§¶
From the Vitamin D, Skin, and Bone Research Laboratory,
Endocrinology Nutrition and Diabetes Section,
§ Departments of Medicine and
Physiology,
Boston University Medical Center, Boston, Massachusetts 02118
 |
ABSTRACT |
We reported previously that the rate of
previtamin D3 (preD3)
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
vitamin D3 isomerization. We found
that a decrease in the hydrophilic interaction of the preD3
with liposomal phospholipids by an esterification of the 3
-hydroxy
of preD3 (previtamin D3-3
-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 3
-acetate, and 25-hydroxy derivatives to
their corresponding vitamin D3 compounds. We also
determined rate constants (k) of preD3
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 |
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
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
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
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
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
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-3 -acetate. B, R = H,
X = H: cZc-previtamin D3; R = H, X = OH: cZc-25-hydroxyprevitamin D3;
R = CH3CO-, X = H:
cZc-previtamin D3-3 -acetate. C,
R = H, X = H: vitamin D3;
R = H, X = OH: 25-hydroxyvitamin
D3; R = CH3CO-;
X = H: vitamin D3-3 -acetate;
D, R = H; X = H: tZc-previtamin
D3; R = H, X = OH: tZc-25-hydroxyprevitamin
D3; R = CH3CO , X = H:
tZc-previtamin D3-3 -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 3 -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.
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EXPERIMENTAL PROCEDURES |
Materials
7-DHC and vitamin D3 were purchased from Sigma.
7-DHC-3
-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 3
-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-3
-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
|
(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:
|
(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 (
max = 260 nm) and its ability to thermally isomerize to 25(OH)D3.
Base-line separation of vitamin D3-3
-acetate,
preD3-3
-acetate, and 7-DHC-3
-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.
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Kinetics of PreD3
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 vitamin
D3 isomerization in isotropic (n-hexane
and DPPC/n-hexane solution) versus
anisotropic microenvironments (human skin and DPPC
liposomes).
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Temperature Dependence on Rate Constants of the Isomerization in
DPPC Liposomes--
The rate constants of preD3
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,
|
(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).
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-3
-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 C3
position by esterification (preD3-3
-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, preD3-3
-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 preD3-3
-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 ( ), 25(OH)preD3 ( ), and
preD3-3 -acetate ( ) to their corresponding vitamin
D3 analogs in n-hexane
(A) and in DPPC liposomes
(B).
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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
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
|
(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 vitamin
D3 isomerization in liposomes. k, rate constant;
n, carbon number of phospholipid chain.
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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
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
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
vitamin D3 isomerization. We found
that in an isotropic solution of DPPC/n-hexane, the rate of
preD3
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
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
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
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 C3
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
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
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
C3
-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 preD3-3
-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 C3
ester group
was less effective than free C3
-OH of preD3
in hydrophilic interaction with polar head groups of liposomal phospholipids.
We speculated that the free 3
-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 3
-OH,
preD3-3
-acetate was unable to form a hydrogen bond with
the carbonyl groups of phospholipids. This weakened the hydrophilic
interactions between preD3-3
-acetate, and membrane
lipids probably reduced the efficiency of stabilizing the
preD3-3
-acetate in its cZc conformer and led to a slower
rate of its isomerization to vitamin D3-3
-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
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 3
-
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
25(OH)D3
isomerization was anticipated to be reduced more markedly than the rate
of preD3-3
-acetate
vitamin
D3-3
-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
vitamin D3,
isomerization was examined.
We first carried out kinetic studies of preD3
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
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
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.
 |
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