Lipid Phase Behaviour

Tsvetkova, N.M., and P.J. Quinn (1994). Compatible solutes modulate membrane lipid phase behaviour. In Temperature Adaptation of Biological Membranes, pp. 49-61, ed. A.R. Cossins. London Portland Press. 

Since the amphiphilic nature is essential for the phase behaviour, systems of small molecules (e.g., lipid water mixtures) and polymeric systems (e.g., homopolymer copolymer blends) share many connnon features. 

In this work we will focus on the use of the cubic phase as a delivery system for oligopeptides - Desmopressin, Lysine Vasopressin, Somatostatin and the Renin inhibitor H214/03. The amino acid sequences of these peptides are given in Table I. The work focuses on the cubic phase as a subcutaneous or intramuscular depot for extended release of peptide drugs, and as a vehicle for peptide uptake in the Gl-tract. Several examples of how the peptide drugs interact with this lipid-water system will be given in terms of phase behaviour, peptide self-diffusion, in vitro and in vivo release kinetics, and the ability of the cubic phase to protect peptides from enzymatic degradation in vitro. Part of this work has been described elsewhere (4-6). 

Sujak, A., K. Strzalka, and W.I. Gruszecki. 2007b. Thermotropic phase behaviour of lipid bilayers containing carotenoid pigment canthaxanthin A differential scanning calorimetry study. Chem. Phys. Lipids 145 1-12. 

It is important for the theoretical understanding of the formation of various topologies that these aggregates have entropic contributions on the scale of the objects, i.e. on a much larger scale than set by the molecules. These cooperative entropic effects should be included in the overall Helmholtz energy, and they are essential to describe the full phase behaviour. It is believed that the mechanical parameters discussed above kc,k and J0, control the phase behaviour, where it is understood that these quantities may, in principle, depend on the overall surfactant (lipid) concentration, i.e. when the membranes are packed to such a density that they strongly interact. 

Monte Carlo may be used to study the lateral distribution of lipid molecules in mixed bilayers. This of course is a very challenging problem, and, to date, the only way to obtain relevant information for this is to reduce the problem to a very simplistic two-dimensional lattice model. In this case, the lipid molecules occupy a given site and can be in one of the predefined number of different states. These pre-assigned states (usually about 10 are taken), are representative conformations of lipids in the gel or in the liquid state. Each state interacts in its own way with the neighbouring molecules (sitting on neighbouring sites). Typically, one is interested in the lateral phase behaviour near the gel-to-liquid phase transition of the bilayer [69,70]. For some recent simulations of mixtures of DMPC and DSPC, see the work of Sugar [71]. 

The link from lipid properties to mechanical properties of the bilayers is now feasible within the SCF approach. The next step is to understand the phase behaviour of the lipid systems. It is likely that large-scale (3D) SCF-type calculations are needed to prove the conjectures in the field that particular values of the Helfrich parameters are needed for processes like vesicle fusion, etc. In this context, it may also be extremely interesting to see what happens with the mechanical parameters when the system is molecularly complex (i.e. when the system contains many different types of molecules). Then there will be some hope that novel and deep insights may be obtained into the very basic questions behind nature s choice for the enormous molecular complexity in membrane systems. 

M. W. De Jager, G. S. Gooris, I. P. Dolbnya, W. Bras, M. Ponec, and J. A. Bouwstra. The phase behaviour of skin lipid mixtures based on synthetic ceramides. Chem. Phys. Lipids 124 123-134 (2003). 

NMR measurements on deuterated phospholipid bilayers and phospho-lipid/eholesterol mixtures, measurements of self-diffusion of phosphatidyleho-lines in lipid bilayers, and finally pressure effeets on the strueture and phase behaviour of model biomembranes eonsisting of phospholipid bilayers with incorporated peptides will be discussed. 

Phosphor- lipide, Hexokinase Triton XI00-propylbenzene, Triton XIOO-toluene Comprehensive investigation of the phase behaviour and the influence on enzyme activity and stability [37,38] [37,38]... 

Lafleur, M. 1998. Phase behaviour of model stratum corneum lipid mixtures An infrared spectroscopy investigation. Can J Chem 76 1500. 

J. E. Staggers, O. Hernell, R. J. Stafford, and M. C. Carey, Physical-chemical behaviour of dietary and biliary lipids during intestinal digestion and absorption. 1. Phase behaviour and aggregation states of model lipid systems patterned after aqueous duodenal contents of healthy adult human beings, Biochemistry 29 2028-2040 (1990). 

Engblom, J., On the Phase Behaviour of Lipids with Respect to Skin Barrier Function, Thesis. Lund University, Sweden, 1996. 

Thewalt, J. et al., Models of stratum corneum intercellular membranes the sphingolipid headgroup is a determinant of phase behaviour in mixed lipid dispersions, Biochem. Biophys. Res. Commun., 188, 1247, 1992. 

Brandenburg, K., Blume, A. Investigations into the thermotropic phase behaviour of natural membranes extracted from Gram-negative bacteria and artificial membrane systems made from lipopolysaccharides and free lipid A. Thermochim Acta 119 (1987) 127-142. 

As shown in the Figure 2, compared to the corresponding homopolymer, the copolymers show a liquid expanded phase depending on the comonomer content. This liquid expanded phase behaviour is not caused by a phase transition in the side chain as found with many natural and synthetic lipids. In this case it can be explained as an entropy driven coiling (B)—uncoiling (A) process of the copolymeric main chain (Frey et al., 1987). 

For this purpose liposomes are used as lipid phase. Unilamellar liposomes are artificial lipid bilayer vesicles. They can be considered as real model bilayer membranes as they ideally consist of a circular bilayer membrane. The hydrophobic acyl chains are assembled in the hydrophobic core of the liposome whereas the hydrophilic head groups point to the water in the inside and outside of the vesicle. Liposomes can be produced from a variety of lipids and from mixtures of lipids. This possibility allows studying the influence of membrane constituents on the partition of solutes. Kramer et al. (1997) studied the influence of the presence of free fatty acids in membranes on the partition behaviour of propranolol. The influence on a-Tocopherol in membranes on the partition behaviour of desipramine has been reported recently (Marenchino et al. 2004) using a liposome model. 

The apparatus used to study the phase behaviour of the lipids in SCCO2 has been described in details by Hammam and Sivik [8]. All experiments were carried out at 90 bar and 40°C. In this work the sapphire cell used was charged with 0.25 g lipid sample and 0.16 g ethanol (99.5 %) and the phase behaviour of this mixture observed visually. The amount of ethanol and lipid sample was chosen so that they would represent the proportions of ethanol, lipids and SCCO2 present in the actual enzyme reaction experiment. 

Phase behaviour of mixtures of ethanol and different lipid samples in SCCO2 at 90 bar and 40 °C... 

The main difference between the lipid samples under investigation is their lipid class composition (Table 1). The unreacted CLO contains nearly only TG while the main difference between Residue A and B was the amount of TG present in the sample. As there was no large difference in the amount of amphiphilic components, i.e. DG and MG, present in the two residues studied, additional MG (Dimodan) was added to one of the residues to investigate the effect of this. The results showed that two phases, a gas phase and a yellow liquid phase, were observed in all sample mixtures studied (Table 1). To study whether intensive mixing of the two phases present would effect their phase behaviour, the sapphire cell was turned up and down a couple of times. This resulted in a hazy gas phase, while the yellow liquid phase remained transparent. However, when the cell was merely swirled around once or twice, exclusively the gas phase in immediate vicinity to the phase boarder became hazy. These results suggest that... 

Hence, the interaction between lipid molecules is very similar in these foam bilayers and it can be supposed that the AF foam bilayers are in the liquid crystalline state within the temperature range studied. This assumption is in agreement with the fact that amniotic fluid contains substantial amount of unsaturated phospholipids, which as known [45], lower considerably the temperature of the chain-melting phase transition. Bearing in mind the similarity of the phase behaviour of a phosphatidylcholine aqueous dispersion and foam bilayers [38-40], it can be supposed that at the temperatures which are important for in vivo systems, the foam bilayers are in the liquid crystalline state. This assumption allows to determine the critical concentration of phosphatidylcholines in amniotic fluid, necessary for formation of a foam bilayer by extrapolation of the Arrhenius dependence of C, for AF foam bilayers to 37°C. Thus, at 37°C C, = 19.9 jxg cm 3 and d, = 1.47. This value of C, at 37°C corresponds to the lower limit (found by other methods [46,47]) of phosphatidylcholine concentration which permits to classify as mature a sample of amniotic fluid. The above value... 

The influence of other food components (especially of low molecular weight, such as lipids, sugars and polyvalent metal ions) and operating conditions, such as high shear forces, high pressure and sufficiently high temperature (e.g., pasteurization and sterilization), on phase behaviour of protein-polysaccharide mixtures remains only partially studied. 

Engblom, J. On the phase behaviour of lipids with respect to skin barrier function. Ph.D. thesis. Department of Food Technology, Lund, Sweden, 1996. 

Lewis RNAH, McElhaney RN. The mesomorphic phase behaviour of lipid bilayers. In The Structure of Biological Membranes. Yeagle PE, ed. 1991. CRC Press, Boca Raton, FL. pp. 73-155. Ipsen JH, Karlstrom G, Mouritsen OG, Wennerstrom H, Zuk-ermann MJ. Phase equilibria in phosphatidylcholine-cholesterol system. Biochim. Biophys. Acta 1987 905 162-172. 

The phosphorescence of trivalent cations (as analogues of Ca ) is also widely used in binding studies. The photobinding of phenothiazine derivatives has been studied for different types of biological membranes. The specificity of binding is low, although general, and can be used to identify and localize membrane proteins. The influence of Ca " and phase behaviour in synaptosomal lipids have been examined by the steady-state fluorescence polarization of A fluorescent probe of the tumour promoter phorbol... 

We begin with the field of lipid-water phase behaviour. 

Besides the asymmetry between monolayers in cytomembranes, two of the more obvious differences between cubic phases and membranes are the unit cell size and the water activity. It has been argued that tire latter must control the topology of the cubic membranes [15], and hence tiiat the cubic membrane structures must be of the reversed type (in the accepted nomenclature of equilibrium phase behaviour discussed in Chapters 4 and 5 type II) rather than normal (type I). All known lipid-water and lipid-protein-water systems that exhibit phases in equilibrium with excess water are of the reversed type. Thus, water activity alone cannot determine the topology of cubic membranes. Cubic phases have recently been observed with very high water activity (75-90 wt.%), in mixtures of lipids [127], in lipid-protein systems [56], in lipid-poloxamer systems [128], and in lipid A and similar lipopolysaccharides [129,130]. 

What are the functions, if any, of the cubic membranes It may be that cubic membranes are but an inevitable self-assembled product of the complex molecular soup of lipids and proteins the result of molecular packing considerations and inter-molecular interactions. This would be in analogy with known phase behaviour in equilibrium systems. Even though this is a very appealing solution to the long and unresolved debate about "non-lamellar" lipids in conjunction with cell membranes, we rather believe that these structural organisations have been chosen to fulfil a purpose (see, e.g. [134] and references therein for current theories, and [4] for a more comprehensive discussion), and the formation cannot be rationalised solely by molecular packing. 

Timms, R.E., Phase Behaviour of Fats and Their Mixture, Prog. Lipid Res. 23 1-38 (1984). 

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