2:1 DPPC/cholesterol

Table III Comparison of Percent of Total Gauche Conformers for Pure DPPC, 2 1 DPPC/Cholesterol, and DPPC/Gramicidin Mixtures.

Pyranine has been used to study the proton dissociation and diffusion dynamics in the aqueous layer of multilamellar phospholipid vesicles [101], There are 3-10 water layers interspacing between the phospholipid membranes of a multilamellar vesicle, and their width gets adjusted by osmotic pressure [102], Pyranine dissolved in these thin layers of DPPC and DPPC+cholesterol multilamellar vesicles were used as a probe for the study. Before the photoreleased proton escapes from the coulombic cage, the probability of a proton excited-anion recombination was found to be higher than in bulk. This was attributed to the diminished water activity in the thin layer. It was found that the effect of local forces on proton diffusion at the timescale of physiological processes is negligible. 

DPPC/Cholesterol Mixtures. To explore this advantage, an obvious starting point is the investigation of cholesterol/DPPC systems. 

Another example worth mentioning is X-ray diffraction studies on amiodarone [80, 81], a drug that accumulates extensively in membranes (see Section 4.4). The effect of cholesterol on membrane structure has also been studied by X-ray diffraction. The results indicated the existence of microdomains in the DPPC-cholesterol mixed ripple phase [82]. 

More recently, MD simulations under constant pressure involving full representation of phospholipids and cholesterol have been carried out. They dealt either with DMPC- [70, 71] or DPPC-cholesterol mixtures [72, 73]. The results of these four simulations were generally in agreement but differed in some aspects. 

In the simulation of Tu et al. [72] with 12.5 mol% cholesterol, the starting configuration was obtained from a simulation of a pure DPPC bilayer composed of 64 DPPC molecules. Four DPPC molecules per leaflet were replaced by cholesterol to generate the DPPC-cholesterol system. The system was simulated for 1.4 ns the area per phosholipid decreased during the first 700 ps of the simulation but then remained stable for the rest of the simulation. As pointed out by the authors, the decrease in the surface area was due to the replacement of the phospholipids by the thinner cholesterol and not to an ordering effect of cholesterol. Accordingly, little effect of cholesterol on the order of the alkyl chains was observed in this simulation, in contradiction to the results of a cholesterol-DPPC simulation performed by Smondyrev and Berkowitz that is described below. 

Fig. 6.11 Schematic representation of initial structures of the DPPC/cho-lesterol and DPPC/cholesterol sulfate membranes. PL phopholipid, CH cholesterol, CS cholesterol sulfate. Left system A, middle system B [73], right DPPC/cholesterol sulfate [75]...

Smondyrev and Berkowitz [73] performed a rather long simulation (2 ns) of a system with a low cholesterol content (ratio of 1 8 or 11 mol%) and of two systems that contained cholesterol at 1 1 molar ratio and differed in the arrangement of the cholesterol molecules (Figure 6.11). In the simulation with low cholesterol content, the area per lipid was about 62 A2, close to that found in a simulation of pure DPPC for the first approximately 800 ps. However, after this time it started to decrease and reached a value of 58.3 A2 after an additional 500 ps, where it remained for the last 750 ps of the simulation. In the simulation with high cholesterol content, the area per DPPC-cholesterol heterodimer surface area showed also a decrease over the simulation time that was approximately exponential (Table 6.7). In system A (Figure 6.11), with a more uniform distribution of cholesterol, the surface area tended to decrease over the full simulation time of 2 ns, whereas for system B a stable and somewhat lower value was obtained after 1 ns. Although the authors concluded that system A, with more uniformly distributed cholesterol, might be trapped in a metastable state, this system was used in a later publication for comparison of the effects caused by cholesterol sulfate (see below). 

Tab. 6.8 Average number of hydrogen bonds per DPPC or cholesterol oxygen, formed with water molecules reported from several simulations. Data for DPPC and DPPC-cholesterol are taken from ref. 73 and data for DMPC-cholesterol sulfate are from ref. 75. Data for DMPC-cholesterol are taken from ref. 71 and data for pure DM PC are taken from ref. 74 and 106.

Oxygens Pure DPPC DPPC-cholesterol 11 mol% 50 mol%a DPPC-cholesterol sulfate 50 mol% Pure DM PC DM PC cholesterol 22 mol%... 

Some commonly used lipids include DPPC, cholesterol, and phosphatidylcholine (U,15). 

Vibrational spectroscopy also shows interactions of polyene antibiotic ion channels nystatin and amphotericin B with phospholipid bilayers (Bunow and Lewin, 1977a Iqbal and Weidekamm, 1979 Van de Ven et al., 1984). In particular, Fourier Transform Raman spectroscopy demonstrates that at high temperature, the amphotericin A complex of DPPC/cholesterol is more ordered, whereas the amphotericin B complex is as ordered as the pure lipid/cholesterol system. In the low temperature phase and in the presence of the sterol-antibiotic complex, the bilayers were suggested to be in the interdigitated state (Levin and Neil Lewis, 1990). 

The DPPC/cholesterol phase diagram in Fig. 5h contains a critical point. It is related to the existence of a peculiar, liquid ordered lo) phase in the mixtures, which is believed to be the prototype of the lipid rafts (see Lipid rafts). 

We note here that systematic studies of the melting transition of dry or nearly dry phospholipids bilayers (e.g., vesicles) have been scarce. While there is an abundant experimental and theoretical literature concerning the structure and properties of bilayers in water, less is known about their behavior when water is removed. We have therefore initiated a systematic experimental study of the gel-liquid crystal transition of pure DPPC and DPPC-cholesterol vesicles freeze-dried with and without disaccharides and oxyanion-disaccharide complexes. Some of our results to date are shown in Figure 9.3. 

Figure 41.1 shows the gel-to-liquid crystalline phase transition temperatures (Tm) of DPPC-cholesterol mixtures as a function of the cholesterol-lipid molar ratio. The Tm of fully hydrated DPPC is 42°C (Crowe and Crowe, 1988 Vist and Davis, 1990 McMullen et al., 1993 Ohtake et al., 2004). Upon the addition of cholesterol, the transition enthalpy decreases continuously imtil it is no longer observable at 50 mol% cholesterol. The disappearance of the melting transition has been attributed to strong interactions between cholesterol and DPPC (McCoimell, 2003). Upon dehydration, the Tm for DPPC increases from 42 to 105°C (Crowe and Crowe, 1988 Ohtake et al., 2004). This Tm increase is caused by the reduction in the spacing between the phospholipids, which allows for increased van der Waals interactions between the lipid hydrocarbon chains (Koster et al., 1994). Between 10 and 70 mol% cholesterol, two endothermic transitions are observed, both lower than the Tm of the pure phospholipid (Figure 41.1). High-sensitivity DSC studies on fully hydrated DPPC-cholesterol systems reported endotherms consisting of two components, suggesting the existence of domains enriched/depleted in cholesterol (Vist and Davis, 1990 McMullen et al., 1993). The two peaks present in our freeze-dried systems also suggest the...

Phase-transition temperature-composition diagram for the fully hydrated and freeze-dried DPPC-cholesterol liposomes. 

In this work, we have analyzed the phase behavior of various freeze-dried mixtures of DPPE, DPPC, and cholesterol and have examined the effects of trehalose addition to these liposomes. Generally, dehydration leads to increase in transition temperature of the phospholipids and also to phase separation. Addition of trehalose, however, can prevent the increase in transition temperature and phase separation freeze-dried DPPC-cholesterol liposomes exhibit only one transition and their retention capability increases by more than 40%. Further studies on the phase separation and stability of multicomponent model membranes will be required to understand better its relation to the survival of cells to freeze-drying procedures. 

Effect of cholesterol addition on the retention of calcein by DPPC-cholesterol liposomes. 

Fig. 16. Isothermal compressibility data of DPPC-cholesterol mixtures as a function of cholesterol concentration and pressure at T= 50 °C [98].

Fig. 17. Pressure dependence of the steady-state fluorescence anisotropy r g of TMA-DPH in DPPC/cholesterol unilamellar vesicles at different sterol concentrations (7 = 50 °C).

Fig. 3. Temperature dependence of the maximum wave number of the CHj asymmetric stretching vibrations in (a) DPPC-cholesterol and (b) DMPC-cholesterol dispersions at the molar ratios indicated. The temperature dependence for the pure lipids is also given [48].

Perhaps the most detailed study of DPPC-cholesterol mixtures by DSC is the one performed by McMullen and McElhaney, whose phase diagram is reproduced in... 

Figure 4.4 Temperature-composition diagram of DPPC-cholesterol in excess water. [Reproduced from T.P.W. McMullen and R.N. McELhaney (1995) Biochim. Biophys. Acta 1234 90-98, with permission]...

Figure 4.5 Sharp and broad components in a DSC thermogram. The sample corresponds to DPPC-cholesterol (94 6 mole ratio) in excess water. [Reproduced from McMullen and McElhaney (1995), with permission]...

Similar DSC studies have been carried out with a related binary mixture, namely sphingomyelin/cholesterol. Different sphingomyelins (SM) vary in melting temperature, but all of them appear to have a gel-fluid transition. AT-palmitoyl SM, the main component of egg SM has a transition at 41 °C. Its mixtures with cholesterol behave similarly to the DPPC/cholesterol system, in that (a) cholesterol causes the endotherm to broaden and its enthalpy to decrease without marked changes in the transition temperature (b) the transition is abolished at or above 50 mol percent cholesterol, and (c) mixtures containing up to 20 mol per cent sterol can be deconvolved into a sharp... 

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