Vitamin D 2 at high and low concentrations exert opposing effects on molecular order and dynamics of dipalmitoyl phosphatidylcholine membranes

Fourier Transform Infrared spectroscopic studies show that low concentrations of vitamin D 2 (1 and 3 mol %) does not induce significant change in the overall shape of the thermotropic profile of dipalmitoyl phosphatidylcholine (DPPC) membrane. In contrast, at higher concentrations of vitamin D 2 (9 and 12 mol %), the phase transition shifts to lower temperatures and a significant broadening in the phase transition curve is also observed. Low concentration of vitamin D 2 decreases the frequency of the CH2 stretching mode, implying an ordering effect, whilst high concentration of vitamin D 2 disorders the system. Furthermore, at low and high concentrations, vitamin D 2 causes opposing effect on membrane dynamics. It decreases the bandwidth of the CH 2 stretching modes at low concentrations while increasing it at high concentrations. We have also observed different actions of vitamin D 2 at low and high concentrations in the deep interior and interfacial region of the membrane, by monitoring the frequency of the CH 3 stretching band and C=O stretching bands, respectively.


Introduction
Vitamin D 2 (ergocalciferol) is a lipid soluble vitamin, which does not occur naturally.It is obtained by irradiation of the provitamin ergosterol from yeast which have ergosterol as their sterol component.It is inactive and exerts its biological activity as a consequence of further metabolism.It plays an important role in calcium and phosphorus metabolism, in immune function and cell differentiation [1] and recently it has been proposed as a membrane antioxidant since it has the ability to inhibit iron-dependent lipid peroxidation in liposomes [2].Although several studies were carried out to investigate the metabolism and role of vitamin D in the treatment and prevention of several diseases [2][3][4][5][6][7][8][9], the molecular mechanism behind such diverse function of vitamin D 2 has not taken much attention as yet.In order to better understand the function of vitamin D 2 at the molecular level, it is important to study its interaction with membrane components and specifically with lipids.To achieve this, detailed concentration and temperature dependent studies using spectroscopic and calorimetric techniques are essential.In the present study, we have addressed these questions by investigating the interaction of vitamin D 2 with model membranes composed of dipalmitoyl-phosphotidylcholine liposomes.The interactions have been monitored using two non-invasive techniques, namely Fourier transform infrared (FTIR) spectroscopy, and differential scanning calorimetry (DSC).The combination of these techniques allowed us to obtain a detailed picture of the effect of vitamin D 2 on the phospholipid phase behaviour, structure and dynamics which, to the best of our knowledge, has not been reported in the literature.

Materials and methods
Ergocalciferol and DPPC were purchased from Sigma (St. Louis, MO) and were used without further purification.
For infrared measurements, pure phospholipid multilamellar liposomes were prepared according to the procedure reported in Severcan and Cannistraro [10], but with reduced amount (80%) of hydration [11].Infrared spectra were obtained using a BOMEM-157 FT-IR spectrometer.20 µl of samples were placed between CaF 2 windows with the cell thickness of 12 µm.Interferograms were averaged for 100 scans at 2 cm −1 resolution.Temperature was regulated by a Unicam Specac digital temperature controller unit.The samples were incubated for 10 minutes at each temperature before data acquisition.
The lipid mixture for the DSC measurements were prepared according to the same procedure for infrared study, however, this time thin films obtained from 1 mg of phospholipid were hydrated by adding 0.5 ml of double distilled deionized water.A Micro Cal VP-DSC instrument was used with a heating rate of 1 • C/min.Samples were degassed under reduced pressure prior to data collection.Analysis of the DSC data was carried out with the software Origin (Microcal Inc., Northamton, MA).The raw DSC curves were processed using standard procedures [12].Each transition was analyzed independently.A cubic splines interpolation was used for baseline correction [13].

FT-IR studies
The infrared spectra of lipids have been studied in detail and most bands have been assigned [14][15][16].Various kinds of information can be derived from these bands.Frequency shifts in different regions or changes in the widths of the corresponding peaks can be used to extract information about various physicochemical processes taking place.For example, the frequencies of the CH 2 stretching bands of acyl chains depend on the degree of conformational disorder and hence can be used to monitor the average trans/gauche isomerization in the systems.The shifts to higher wavenumbers correspond to an increase in number of gauche conformers [14][15][16] and bandwidth gives dynamic information about the system [16].
The infrared spectra of DPPC multilamellar liposomes, both pure and containing different concentration of vitamin D 2 were investigated as a function of temperature.The C-H stretching modes, the CH 2 scissoring mode and the C=O stretching mode were considered.The results presented here refer to the effect of vitamin D 2 on phospholipid membrane structure by monitoring these vibrations corresponding to DPPC.
Figure 1 shows the temperature dependence of the frequency of the CH 2 symmetric stretching modes of DPPC liposomes in the presence and absence of different concentrations of vitamin D 2 .The frequency values at temperatures below 32 • C are characteristic of conformationally highly ordered acyl chains with a high content of trans isomers as found in solid hydrocarbons, whereas, the values at temperatures above 45 • C are characteristic of conformationally disordered acyl chains with a high content of gauche conformers such as those found in liquid hydrocarbon.The abrupt shift in the peak frequency of the CH 2 stretching modes of DPPC which takes place during the main endothermic phase transition (∼41 • C) has been associated with the change from all trans to gauche conformers [16].As seen from the figure with the addition of 1 and 3 mol % vitamin D 2 , the shape of the phase transition curve does not change and no significant shift for the midpoint temperature of phase transition curve is observed.The effect of high concentration of vitamin D 2 (9 and 12 mol %) on the thermotropic phase transition is very different than those of the lower vitamin D 2 concentration.The gel to liquid crystalline phase transition is completely abolished and the curve significantly broadens implying the loss of cooperativity between the lipid chains of DPPC.
As seen from Fig. 1, the effect of vitamin D 2 on the frequency of the CH 2 stretching mode varies also for different vitamin D 2 concentrations.Inclusion of 1 and 3 mol % vitamin D 2 decreases the frequency, whereas an abrupt increase in the frequency is observed with the addition of 9 mol % vitamin D 2 .The frequency is still further above the DPPC value in the presence of 12 mol % vitamin D 2 although the effect is less profound in the liquid crystalline phase.
Figure 2 shows the temperature dependence of the bandwidth of the symmetric stretching band of DPPC in the absence and presence of vitamin D 2 .Bandwidth was measured at 0.75 × peak height position.Qualitatively similar results are also obtained at 0.50 × peak height position.As illustrated in the figure, low (1 and 3 mol % ) and high (9 and 12 mol % ) vitamin D 2 concentrations induce different effects.At any given temperature, the bandwidth decreases for low vitamin D 2 concentrations, but a significant increase is observed at high concentrations of vitamin D 2 both in the gel and liquid crystalline phases.
The band at 2956 cm −1 results from the asymmetric stretching vibrations of the terminal methyl group of the palmitoyl chains and it provides a monitor of the center of the lipid bilayer [16].Temperature dependent changes in the frequency of this CH 3 asymmetric stretching band of DPPC vesicles, in the presence and absence of vitamin D 2 , are presented in Fig. 3.The decrease in the frequency, with   increasing temperature, reflects a decrease in the vibrational freedom of the acyl chains in the central region of the lipid bilayer [16].Addition of 1 and 3 mol % vitamin D 2 lowers the frequency of the CH 3 asymmetric stretching band.In contrast, a dramatic increase in the frequency is observed with 9 mol % vitamin D 2 , indicating that vitamin D 2 increases the dynamics of the deep interior of the bilayer at high vitamin concentrations.The effect of 12 mol % vitamin D 2 is less pronounced than 9 mol % although it still increases the frequency.
In order to get information about the polar part of the membrane, the C=O stretching band at 1730 cm −1 and PO − 2 antisymmetric stretching band at 1260 cm −1 have been investigated for both phases.Temperature dependence of the frequency of the composite band resulting from the C=O stretching modes of the palmitoyl sn-1 and sn-2 ester groups of pure DPPC and DPPC multibilayers containing different concentrations of vitamin D 2 are shown in Fig. 4. As seen from the figure, addition of low concentration of vitamin D 2 causes a large shift in the band maximum to higher frequencies and this effect decreases at high vitamin D 2 concentrations.A dramatic decrease in the frequency, in comparison to DPPC alone, is observed in the presence of high vitamin D 2 concentrations, in the gel phase.Since the C=O stretching band directly reflects the changes in the lipid head groups, reorientation of lipid head group may be reflected as a frequency increase.This may indicate the existence of free carbonyl groups which does not make hydrogen bonding with the oxygen of carbonyl groups in the presence of vitamin D 2 .The frequency of the 1260 cm −1 band, corresponding to the PO − 2 antisymmetric stretching vibration, shifts slightly to higher values with the addition of low and high concentrations of vitamin D 2 into DPPC liposomes (not shown).

DSC studies
The DSC curves for pure DPPC liposomes and DPPC/ vitamin D 2 systems are shown in Fig. 5.The trace for pure DPPC liposomes show two transition peaks: a small transition at 34.72 • C corresponding to the pretransition and the sharp chain-melting transition occurring at 41.06 • C [17].With the addition of 1 mol % vitamin D 2 , the pretransition disappears and the intensity of the main peak decreases.A simultaneous broadening and shift of the main peak to a lower temperature is also observed.As vitamin D 2 concentration is raised, a further broadening and a shift of the main transition towards lower temperatures is observed.At 9 mol % vitamin D 2 , the main peak is split into two components.These peaks are more clearly seen at 12 mol % vitamin D 2 and occur at 37.73 • C and 40.79 • C.

Discussion
In the present work the effect of vitamin D 2 on the phase transitions profile of DPPC membranes was precisely monitored using two non-perturbing techniques namely infrared spectroscopy and differential scanning calorimetry.Infrared spectroscopy enables one to monitor these transitions via a number of functional groups and to observe differences in their thermotropic behaviour.In this respect we were able to investigate the effect of vitamin D 2 on the hydrophilic part of the membrane by monitoring the C=O stretching band and hydrophobic part of the membrane by monitoring the C-H stretching region.
Our FTIR and DSC studies reveal that addition of vitamin D 2 into the DPPC membrane system eliminates the pretransition, and the main phase transition is shifted to lower temperatures with increasing vitamin D 2 concentrations.This is in agreement with a previous DSC study, which investigated the interaction of vitamin D 3 with phospholipid membranes [17].
As seen throughout the paper, high and low concentration of vitamin D 2 exact opposing effects on DPPC membrane order (acyl chain flexibility) and dynamics.As observed through the analysis of the CH 2 stretching vibration, the inclusion of low vitamin D 2 concentration decreases the number of gauche conformers and membrane dynamics, whilst the inclusion of 9 and 12 mol % vitamin D 2 significantly increases the conformational disorder and dynamics of the lipids.This type of opposing effect between low and high concentrations of vitamin D 2 has also been observed for the CH 3 asymmetric and C=O stretching vibrations.At low vitamin D 2 concentration the C=O stretching vibration of the lipid ester groups shift to a higher frequency which reflects a reduction in hydrogen bonding within the carbonyl groups.It is possible that vitamin D 2 molecules displace some H 2 O molecules from the interfacial region resulting in an increase in the number of free carbonyl groups.In contrast, high vitamin D 2 concentrations cause a shift of the 1730 cm −1 band to a lower frequency.This shift may indicate a greater hydration of the carbonyl groups resulting in an increase in the number of H-bonded carbonyls.It may also reflect an increase in H-bonding between the carbonyl groups and the hydroxyl groups of the vitamin D 2 .The phosphate (PO − 2 ) band is highly sensitive to changes in hydration [18].The lack of any significant shift in the position of this band in the presence of vitamin D 2 rules out any major changes in the hydration of the phosphate groups.It also suggests that vitamin D 2 molecules are unlikely to be involved in any major interaction with the phosphate groups.
It is difficult to explain the precise reason for the different behaviour of vitamin D 2 at low and high concentrations.One possible explanation is that at low concentrations vitamin D 2 molecules are unable to disturb the tight packing of the lipid acyl chains.Hence the co-operative transition is still observed.The ordering effect observed at the low concentration can be attributed to strong hydrophobic interaction between vitamin D 2 and DPPC acyl-chain without leading to a loss of the cooperative transition.However, at higher concentrations of vitamin D 2 , interaction between vitamin D 2 molecules and phospholipid acyl chain may become more dominant than the interaction between the phospholipid molecules.As a result of this, vitamin D 2 molecules may form complexes with phospholipids, as reported previously for vitamin E/phospholipid systems [19], thereby abolishing the characteristic phase transition of DPPC.This strong interaction between the vitamin D 2 molecules and DPPC may also cause disordering and destabilizing effect on the membrane.
The peak observed in the DSC curve at 41 • C decreases in intensity, broadens and shifts to a lower temperature (see Fig. 5) as the vitamin D 2 concentration is raised.This may suggest the co-existence of more than one domain.If these domains are sufficiently large, the exchange of lipids between the domains can not be resolved and the DSC curve will be a superimposition of more than one component.As vitamin D 2 concentration is increased up to 9 mol %, more than one peak appears in the calorimetric profile indicating that lateral phase separation of lipids are indeed occurring probably producing phases with different ratios of vitamin D 2 and phospholipid.In the presence of 12 mol % vitamin D 2 , the main peak is clearly resolved into two components with phase transition temperatures of 37.73 • C and 40.79 • C. The peak observed at higher temperature is virtually identical in position to that observed with pure DPPC.Previous studies on binary mixture of phospholipids with cholesterol [20][21][22][23] and vitamin E [10,19] reported the existence of two different phases as cholesterol/vitamin E-poor and cholesterol/vitamin Erich domains, which have different dynamics.We suggest that the peak observed at a higher temperature in the DSC curve in our study most likely represents the transition of the vitamin D 2 poor domain, whilst the peak at lower temperatures might arise from the vitamin D 2 rich domains.

Conclusion
The present study demonstrates that vitamin D 2 interacts with phospholipid membranes and modulates the physical properties of the lipid bilayer.On the basis of the results obtained in this study we propose a model where vitamin D 2 is located in both monolayers, is arranged with the phenolic hydroxyl group located near the carbonyl moiety of the lipid interfacial region.The molecule is mainly localized in the cooperativity region of the bilayer and interacts with the methylene and terminal methyl groups causing significant changes in the physical parameters of the membrane.The stabilizing effect induced by vitamin D 2 at low concentrations suggests that it may exert its reported antioxidant action by a similar membrane stabilizing effect which has been observed for other antioxidants [10,19,24,25].Concentration dependent modulation of phospholipid membrane by vitamin D 2 could be important for its therapeutic properties.

Fig. 1 .
Fig. 1.Temperature dependence of the peak frequency of the CH2 symmetric stretching mode for pure DPPC and DPPC containing varying concentrations of vitamin D2.

Fig. 2 .
Fig. 2. Temperature dependence of the bandwidth of the CH2 antisymmetric stretching mode for pure DPPC and DPPC containing varying concentrations of vitamin D2.

Fig. 3 .
Fig. 3. Temperature dependence of the peak frequency of the CH3 asymmetric stretching mode for pure DPPC and DPPC containing varying concentrations of vitamin D2.

Fig. 4 .
Fig. 4. Temperature dependence of the peak frequency of the C=O stretching mode for pure DPPC and DPPC containing varying concentrations of vitamin D2.

Fig. 5 .
Fig. 5.The DSC calorimetric curves for pure DPPC and DPPC containing varying concentrations of vitamin D2.