Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1_c phosphatidylcholine and 16:0/18:1_c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using ³¹P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with ³¹P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H_II phase, 16:0/15:1_c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.     

Spin-label characterisation of the lamellar-to-hexagonal (HII) phase transition in egg phosphatidylethanolamine aqueous dispersions

The thermotropic behaviour of egg yolk phosphatidylethanolamine dispersions in excess aqueous phase has been investigated by spin label electron spin resonance spectroscopy and differential thermal analysis. Phosphatidylethanolamine isomers spin-labelled at six different positions along the acyl chain, and steroid spin labels, indicate both gel-fluid lamellar and lamellar-reverse hexagonal (HII) phase transitions, in agreement with complementary calorimetric studies. Analysis of spin label data shows that the transition to the HII phase is accompanied by an increase in conformational freedom of the acyl chain, more pronounced towards the methyl terminus, and representing an increase in the population of gauche isomers which can only be accommodated by a transition to the non-bilayer phase. Raising the bulk pH to, and above, pH 8.5 results in stabilisation of the bilayer phase and no transition to the HII phase is observed. The phosphatidylethanolamine spin labels also indicate a polarity profile which is characteristic of each phase.     

Cholesterol favors phase separation of sphingomyelin

The phase behavior of mixed lipid dispersions representing the inner leaflet of the cell membrane has been characterized by X-ray diffraction. Aqueous dispersions of phosphatidylethanolamine:phosphatidylserine (4:1 mole/mole) have a heterogeneous structure comprising an inverted hexagonal phase H(II) and a lamellar phase. Both phases coexist in the temperature range 20-45 degrees C. The fluid-to-gel mid-transition temperature of the lamellar phase assigned to phosphatidylserine is decreased from 27 to 24 degrees C in the presence of calcium. Addition of sphingomyelin to phosphatidylethanolamine/phosphatidylserine prevents phase separation of the hexagonal H(II) phase of phosphatidylethanolamine but the ternary mixture phase separates into two lamellar phases of periodcity 6.2 and 5.6 nm, respectively. The 6.2-nm periodicity is assigned to the gel phase enriched in sphingomyelin of molecular species comprising predominantly long saturated hydrocarbon chains because it undergoes a gel-to-fluid phase transition above 40 degrees C. The coexisting fluid phase we assign to phosphatidylethanolamine and phosphatidylserine and low melting point molecular species of sphingomyelin which suppresses the tendency of phosphatidylethanolamine to phase-separate into hexagonal H(II) structure. There is evidence for considerable hysteresis in the separation of lamellar fluid and gel phases during cooling. The addition of cholesterol prevents phase separation of the gel phase of high melting point sphingomyelin in mixtures with phosphatidylserine and phosphatidylethanolamine. In the quaternary mixture the lamellar fluid phase, however, is phase separated into two lamellar phases of periodicities of 6.3 and 5.6 nm (20 degrees C), respectively. The lamellar phase of periodicity 5.6 nm is assigned to a phase enriched in aminoglycerophospholipids and the periodicity 6.3 nm to a liquid-ordered phase formed from cholesterol and high melting point molecular species of sphingomyelin characterized previously by ESR. Substituting 7-dehydrocholesterol for cholesterol did not result in evidence for lamellar phase separation in the mixture within the temperature range 20-40 degrees C. The specificity of cholesterol in creation of liquid-ordered lamellar phase is inferred.     

Acid- and calcium-induced structural changes in phosphatidylethanolamine membranes stabilized by cholesteryl hemisuccinate

The membrane stabilization effect of cholesteryl hemisuccinate (CHEMS) and the sensitivity of the CHEMS-phosphatidylethanolamine membranes to protons and calcium ions were studied by differential scanning calorimetry, freeze-fracture electron microscopy, and 31P NMR. (1) At neutral pH, the addition of 8 mol % CHEMS to transesterified egg phosphatidylethanolamine (TPE) raised the lamellar-hexagonal transition temperature of TPE by 11 degrees C. Stable bilayer vesicles were formed when the incorporated CHEMS exceeded 20 mol %. (2) At a pH below 5.5, the protonation of CHEMS enhanced the formation of the hexagonal phase (HII) of TPE. At 25 mol % CHEMS the bilayer-hexagonal transition temperature was lowered by 30 degrees C at pH 4.5. (3) The endothermic acid-induced hexagonal hexagonal transition of TPE-CHEMS was suppressed at 35 mol % CHEMS. However, 31P NMR and electron microscopy indicated that a lamellar-hexagonal transition still occurred at this composition. (4) The main transition of TPE was not affected by the protonation of the incorporated CHEMS, indicating that no macroscopic phase separation occurred in TPE-CHEMS mixtures at low pH. (5) In contrast to the HII-promoting effect of H+, the neutralization of the negative charge on TPE-CHEMS by Ca2+ resulted in aggregates that remained in the lamellar structure even at the hexagonal transition temperature of TPE. It is suggested that calcium might form a complex between CHEMS in apposed bilayers. These results are related to the possible biological function of acidic cholesterol esters in biomembranes.     

Differential scanning calorimetry of thermotropic phase transitions in vitaminylated lipids: aqueous dispersions of N-biotinyl phosphatidylethanolamines.

The thermotropic phase behavior of a homologous series of saturated diacyl phosphatidylethanolamines in which the headgroup is N-derivatized with biotin has been investigated by differential scanning calorimetry. In 1 M NaCl, derivatives with acyl chainlengths from C(12:0) to C(20:0) all exhibit sharp chain-melting phase transitions, which are reversible with a hysteresis of 1.5 degrees or less, except for the C(12:0) lipid which has a transition temperature below 0 degree C. The transition enthalpy and the transition entropy depend approximately linearly on the lipid chainlength, with incremental values per CH2 group that are very similar to those obtained for the corresponding underivatized phosphatidylethanolamines in aqueous dispersion. The chainlength-independent contribution to the transition enthalpy is significantly smaller than that for the underivatized phosphatidylethanolamines, and that for the transition entropy is much smaller; the latter suggesting that the N-biotinylated phosphatidylethanolamine headgroups are differently hydrated from those of the underivatized lipids. The gel-to-fluid phase transition temperatures of the N-biotinylated lipids are lower than those of the parent phosphatidylethanolamines, and their chainlength dependence conforms well with that predicted by assuming that the transition enthalpy and entropy are linearly dependent on chainlength. Although the chain-melting phase behavior is generally similar to that of the parent phosphatidylethanolamines, the gel phases (and the fluid phases in the case of chainlengths C(12:0) to C(16:0)) have a different lyotropic structure in the two cases, and this is reflected in the chainlength-independent contributions to the thermodynamic parameters. In the absence of salt, the thermotropic phase behavior of aqueous dispersions of the N-biotinyl phosphatidylethanolamines is considerably more complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the stereochemistry of the hydroxyl group of cholesterol and the formation of nonbilayer structures in phosphatidylethanolamines

The phase behavior of mixtures of cholesterol or epicholesterol with phosphatidylethanolamine was studied by differential scanning calorimetry and by X-ray diffraction. Discrete domains of cholesterol are detected by X-ray diffraction in the L alpha phase of phosphatidylethanolamine from egg yolk and synthetic dielaidoylphosphatidylethanolamine beginning at mole fractions of 0.35-0.4 cholesterol. Separate domains of crystalline epicholesterol can also be detected in the L alpha phase of dielaidoylphosphatidylethanolamine by X-ray diffraction at as little as 0.16 mole fraction of epicholesterol. This is a result of poor miscibility of the epicholesterol with dielaidoylphosphatidylethanolamine. Epicholesterol does not alter the L beta----L alpha transition or bilayer spacing. Epicholesterol also has little effect on the diameter of the cylinders in the hexagonal phase. Formation of the inverted hexagonal phase is facilitated by addition of small amounts of cholesterol (mole fraction less than 0.2) in both egg phosphatidylethanolamine and dielaidoylphosphatidylethanolamine. However, at higher mole fractions of cholesterol, the stability of the liquid-crystalline phase is found to increase markedly for dielaidoylphosphatidylethanolamine but not for egg phosphatidylethanolamine, indicating the importance of the structure of the acyl chains in controlling the relative stability of the lamellar and nonlamellar phases in these systems. In contrast to cholesterol, epicholesterol markedly lowers the L alpha----HII phase transition temperature at low mole fraction of sterol. This result demonstrates the importance of the orientation and motional properties of an additive in determining the L alpha----HII transition temperature.     

The inverted hexagonal phase is more sensitive to hydroperoxidation than the multilamellar phase in phosphatidylcholine and phosphatidylethanolamine aqueous dispersions.

The effect of phase behaviour (hexagonal II phase and lamellar phase) on the peroxidation of membrane phospholipids has been investigated in dilinoleoyl phosphatidylcholine (DLPC)/dilinoleoyl phosphatidylethanolamine (DLPE) aqueous dispersions. Peroxidation was initiated with a water-soluble radical inducer 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPN). The phospholipid morphology was monitored by 31P-nuclear magnetic resonance (NMR). Phospholipid hydroperoxides (PCOOH and PEOOH) were determined by chemiluminescence high-performance liquid chromatography (CL-HPLC). In pH-induced phase transition systems, DLPE in the bilayer state was much less oxidized than in the hexagonal II state. In composition-induced phase transition systems, the formation of total hydroperoxides and the consumption of alpha-tocopherol in the hexagonal II phase were greater than in the bilayer phase. These data suggest that the hexagonal II phase is more sensitive to hydroperoxidation than the bilayer phase in phospholipid aqueous dispersions.     

Effect of fatty acyl chain length and structure on the lamellar gel to liquid-crystalline and lamellar to reversed hexagonal phase transitions of aqueous phosphatidylethanolamine dispersions

The lamellar gel/liquid-crystalline and the lamellar liquid-crystalline/reversed hexagonal phase transitions of aqueous dispersions of a number of synthetic phosphatidylethanolamines containing linear saturated, branched chain, and alicyclic fatty acyl chains of varying length were studied by differential scanning calorimetry, 31P nuclear magnetic resonance spectroscopy, and X-ray diffraction. For any given homologous series of phosphatidylethanolamines containing a single chemical class of fatty acids, the lamellar gel/liquid-crystalline phase transition temperature increases and the lamellar liquid-crystalline/reversed hexagonal phase transition temperature decreases with increases in hydrocarbon chain length. For a series of phosphatidylethanolamines of the same hydrocarbon chain length but with different chemical structures, both the lamellar gel/liquid-crystalline and the lamellar liquid-crystalline/reversed hexagonal phase transition temperatures vary markedly and in the same direction. In particular, at comparable effective hydrocarbon chain lengths, both the lamellar gel/liquid-crystalline and the lamellar liquid-crystalline/reversed hexagonal phase transition temperatures vary in parallel, such that the temperature difference between these two phase transitions is nearly constant. Moreover, at comparable effective acyl chain lengths, the d spacings of the lamellar liquid-crystalline phases and of the inverted hexagonal phases are all similar, implying that the thickness of the phosphatidylethanolamine bilayers at the onset of the lamellar liquid-crystalline/reversed hexagonal phase transition and the diameter of the water-filled cylinders formed at the completion of this phase transition are comparable and independent of the chemical structure of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calorimetric and spectroscopic studies of the polymorphic phase behavior of a homologous series of n-saturated 1,2-diacyl phosphatidylethanolamines.

The polymorphic phase behavior of a homologous series of n-saturated 1,2-diacyl phosphatidylethanolamines was investigated by differential scanning calorimetry, 31P-nuclear magnetic resonance, and Fourier transform infrared spectroscopy. Upon heating, aqueous dispersions of dried samples of the short- and medium-chain homologues (n < or = 17) exhibit single, highly energetic transitions from a dry, crystalline form to the fully hydrated, liquid-crystalline bilayer at temperatures higher than the lamellar gel-liquid-crystalline phase transition exhibited by fully hydrated samples. In contrast, the longer chain homologues (n > or = 18) first exhibit a transition from a dehydrated solid form to the hydrated L beta gel phase followed by the gel-liquid-crystalline phase transition normally observed with fully hydrated samples. The fully hydrated, aqueous dispersions of these lipids all exhibit reversible, fairly energetic gel-liquid-crystalline transitions at temperatures that are significantly higher than those of the corresponding phosphatidylcholines. In addition, at still higher temperatures, the longer chain members of this series (n > or = 16) exhibit weakly energetic transitions from the lamellar phase to an inverted nonlamellar phase. Upon appropriate incubation at low temperatures, aqueous dispersions of the shorter chain members of this homologous series (n < or = 16) form a highly ordered crystal-like phase that, upon heating, converts directly to the liquid-crystalline phase at the same temperature as do the aqueous dispersions of the dried lipid. The spectroscopic data indicate that unlike the n-saturated diacyl phosphatidylcholines, the stable crystal-like phases of this series of phosphatidylethanolamines describe an isostructural series in which the hydrocarbon chains are packed in an orthorhombic subcell and the headgroup and polar/apolar interfacial regions of the bilayer are effectively immobilized and substantially dehydrated. Our results suggest that many of the differences between the properties of these phosphatidylethanolamine bilayers and their phosphatidylcholine counterparts can be rationalized on the basis of stronger intermolecular interactions in the headgroup and interfacial regions of the phosphatidylethanolamine bilayers. These are probably the result of differences in the hydration and hydrogen bonding interactions involving the phosphorylethanolamine headgroup and moieties in the polar/apolar interfacial regions of phosphatidylethanolamine bilayers.     

Modulation of membrane structure by Ca2+ and dibucaine as detected by 31P NMR

The polymorphic phase behaviour of model membrane systems consisting of 20 mol% bovine brain phosphatidylserine and 80 mol% egg yolk phosphatidylethanolamine has been examined employing 31P NMR techniques. It is shown that the addition of Ca2+ to such systems can trigger isothermal bilayer to hexagonal (HII) phase transitions, and that such effects can be reversed by the subsequent incorporation of the local anaesthetic dibucaine. These results are discussed in terms of a recent model for membrane fusion (Cullis, P.R. and Hope, M.J. (1978) Nature 271, 672--674) and mechanisms of anaesthesia.     

Destabilization of a lipid non-bilayer phase by high pressure

Pressure is found to destabilize the non-bilayer phase with respect to the bilayer in a model lipid system. The lamellar to inverted hexagonal (H₁₁) phase transition of aqueous egg phosphatidylethanolamine is shifted to higher temperatures by hydrostatic pressure. The slope of the increase in transition temperature is constant to beyond 300 bar, and is greater than that seen for other lipid phase transitions. This behavior is consistent with the hypothesis that increasing chain disorder drives the conversion from the bilayer into the hexagonal phase. If this non-bilayer lipid phase is an intermediate in membrane fusion, then pressure should inhibit the process. This may explain the inhibition of chemical transmission at neural synapses by pressure.     

脂质体通透性与脂多型性关系的研究

本文以TPE和TPE/DOPE(1:1.mol:mol)制成包裹荧光分子calcein的脂质体,通过测量荧光强度随扫描温度的变化,探讨了脂质体通透性与脂多型性之间的关系.结果表明,在不发生双层相(L)变成六角形Ⅱ相(H)相转变时,脂质体悬液的荧光强度不增加;当发生该转变时,脂质体悬液的荧光强度开始增加;完成该相转变后,脂质体悬液的荧光强度仍继续增加.据此,我们认为:脂质体的通透性与脂的多型性密切相关,当发生L→HⅡ相转变时,脂质体的通透性增加.由于荧光强度的变化对相变非常敏感,我们建议用测量脂质体荧光强度随温度的变化来监测脂质体稀悬液中脂的多型性.     

An X-ray diffraction study of the effect of alpha-tocopherol on the structure and phase behaviour of bilayers of dimyristoylphosphatidylethanolamine.

The effect of alpha-tocopherol on the thermotropic phase transition behaviour of aqueous dispersions of dimyristoylphosphatidylethanolamine was examined using synchrotron X-ray diffraction methods. The temperature of gel to liquid-crystalline (Lbeta-->Lalpha) phase transition decreases from 49.5 to 44.5 degrees C and temperature range where gel and liquid-crystalline phases coexist increases from 4 to 8 degrees C with increasing concentration of alpha-tocopherol up to 20 mol%. Codispersion of dimyristoylphosphatidylethanolamine containing 2.5 mol% alpha-tocopherol gives similar lamellar diffraction patterns as those of the pure phospholipid both in heating and cooling scans. With 5 mol% alpha-tocopherol in the phospholipid, however, an inverted hexagonal phase is induced which coexists with the lamellar gel phase at temperatures just before transition to liquid-crystalline lamellar phase. The presence of 10 mol% alpha-tocopherol shows a more pronounced inverted hexagonal phase in the lamellar gel phase but, in addition, another non-lamellar phase appears with the lamellar liquid-crystalline phase at higher temperature. This non-lamellar phase coexists with the lamellar liquid-crystalline phase of the pure phospholipid and can be indexed by six diffraction orders to a cubic phase of Pn3m or Pn3 space groups and with a lattice constant of 12.52+/-0.01 nm at 84 degrees C. In mixed aqueous dispersions containing 20 mol% alpha-tocopherol, only inverted hexagonal phase and lamellar phase were observed. The only change seen in the wide-angle scattering region was a transition from sharp symmetrical diffraction peak at 0.43 nm, typical of gel phases, to broad peaks centred at 0.47 nm signifying disordered hydrocarbon chains in all the mixtures examined. Electron density calculations through the lamellar repeat of the gel phase using six orders of reflection indicated no difference in bilayer thickness due to the presence of 10 mol% alpha-tocopherol. The results were interpreted to indicate that alpha-tocopherol is not randomly distributed throughout the phospholipid molecules oriented in bilayer configuration, but it exists either as domains coexisting with gel phase bilayers of pure phospholipid at temperatures lower than Tm or, at higher temperatures, as inverted hexagonal phase consisting of a defined stoichiometry of phospholipid and alpha-tocopherol molecules.     

Influence of local and neutral anaesthetics on the polymorphic phase preferences of egg yolk phosphatidylethanolamine

(1) The polymorphic phase preferences of egg phosphatidylethanolamine have been examined in the presence of normal alcohols and alkanes of varying chain length, as well as charged amine anaesthetics. (2) It is shown that the charged anaesthetics, ethanol and butanol can stabilize a bilayer arrangement for egg phosphatidylethanolamine. In contrast, longer chain ($\text{C}\text{?6}$) normal alcohols and alkanes induce the hexagonal (H_II) phase. (3) The relative potency of local anaesthetics in vitro (chlorpromazine, dibucaine, tetracaine and procaine) is mirrored by their relative ability to stabilize bilayer structure for hydrated egg phosphatidylethanolamine. Further, the aqueous concentrations of anaesthetic required to affect phospholipid polymorphism is sensitive to the lipid composition. For example, the inclusion of 20 mol% egg phosphatidylserine in egg phosphatidylethanolamine dispersions can reduce the aqueous concentrations of dibucaine required to induce appreciable bilayer stabilization effects from 5.0 mM to 0.5 mM. (4) It is suggested that the ability of amphipatic molecules such as anaesthetics to influence phosphatidylethanolamine polymorphism arises from their molecular shape. The possibility that anaesthetic molecules may exert their effects by virtue of this shape property is raised.     

Effects of temperature variation and phenethyl alcohol addition on acyl chain order and lipid organization in Escherichia coli derived membrane systems. A 2H- and 31P-NMR study.

Using 2H- and 31P-NMR techniques the effects of temperature variation and phenethyl alcohol addition were investigated on lipid acyl chain order and on the macroscopic lipid organization of membrane systems derived from cells of the Escherichia coli fatty acid auxotrophic strain K1059, which was grown in the presence of [11,11-2H2]oleic acid. Membranes of intact cells showed a gel to liquid-crystalline phase transition in the range of 4-20 degrees C, which was similar to that observed for the total lipid extract and for the dominant lipid species phosphatidylethanolamine (PE). Phosphatidylglycerol (PG) remained in a fluid bilayer throughout the whole temperature range (4-70 degrees C). At 30 degrees C acyl chain order was highest in PE, followed by the total lipid extract, PG, intact cells, and isolated inner membrane vesicles. Acyl chain order in E. coli PE and PG was much higher than in the corresponding dioleoylphospholipids. E. coli PE was found to maintain a bilayer organization up to about 60 degrees C, whereas in the total lipid extract as well as in intact E. coli cells bilayer destabilization occurred already at about 42 degrees C. It is proposed that the regulation of temperature at which the bilayer-to-non-bilayer transition occurs may be important for membrane functioning in E. coli. Addition of phenethyl alcohol did not affect the macroscopic lipid organization in E. coli cells or in the total lipid extract, but caused a large reduction in chain order of about 70% at 1 mol% of the alcohol in both membrane systems. It is concluded that while both increasing temperature and addition of phenethyl alcohol can affect membrane integrity, in the former case this is due to the induction of non-bilayer lipid structures, whereas in the latter case this is caused by an increase in membrane fluidity.     

The structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine codispersed with a branched-chain phosphatidylcholine

The structure and thermotropic phase behaviour of a fully hydrated binary mixture of dipalmitoylphosphatidylcholine and a branched-chain phosphatidylcholine, 1, 2-di(4-dodecyl-palmitoyl)-sn-glycero-3-phosphocholine, were examined using differential scanning calorimetry, synchrotron X-ray diffraction and freeze-fracture electron microscopy. The branched-chain lipid forms a nonlamellar phase when dispersed alone in aqueous medium. Mixed aqueous dispersions of the two phospholipids containing less than 33 mol% of the branched-chain lipid form lamellar phases over the whole temperature range were studied (4 degrees C to 60 degrees C). When present in proportions greater than 33 mol% it induces a hexagonal phase in mixed aqueous dispersions with dipalmitoylphosphatidylcholine at temperatures above the fluid phase transition. At temperatures below 35 degrees C a hexagonal phase coexists with a gel bilayer phase. The lamellar<-->nonlamellar transition can be explained satisfactorily on the basis of the shape of the molecule expressed in terms of headgroup and chain cross-sectional areas. At temperatures below 35 degrees C macroscopic phase separation of two gel phases takes place. Freeze-fracture electron microscopy revealed that one gel phase consists of bilayers with a highly regular, periodic superstructure (macro-ripples) whereas the other phase forms flat, planar bilayers. The macro-ripple phase appears to represent a relaxation structure required to adapt to the packing constraints imposed by the incorporation of the branched-chain lipid into the dipalmitoylphosphatidylcholine host bilayer. The data suggest that structural changes that take place on cooling the mixed dispersion below the lamellar<-->nonlamellar phase transition temperature cannot be adequately described using the molecular form concept. Instead it is necessary to take into account the detailed molecular form of the guest lipid as well as its physical properties.     

Diacylglycerols, lysolecithin, or hydrocarbons markedly alter the bilayer to hexagonal phase transition temperature of phosphatidylethanolamines

The bilayer to hexagonal phase transition temperatures of dielaidoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylethanolamine are 65.6 and 71.4 degrees C, respectively. Using high-sensitivity differential scanning calorimetry, I have shown that these transition temperatures are extremely sensitive to the presence of small amounts of other lipid components. For example, at a mole fraction of only 0.01, dilinolenin lowers the bilayer to hexagonal phase transition temperature of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine by 8.5 degrees C. Other diacylglycerols have similar effects on this transition temperature, although the degree of unsaturation of the acyl chains has some effect, with distearin being less potent. In comparison, the 20-carbon alkane eicosane lowers this transition temperature by 5 degrees C, while palmitoyl-lysolecithin raises it by 2.5 degrees C. Similar effects of these additives on the bilayer to to hexagonal phase transition temperature are observed with dielaidoylphosphatidylethanolamine. At these concentrations of additive, there is no effect on the gel-state to liquid-crystalline-state transition temperature. The observed shifts in the temperature of the bilayer to the hexagonal phase transition can be qualitatively interpreted in terms of the effects of these additives on the hydrophilic surface area and on the hydrophobic volume. Substances expanding the hydrophobic domain promote hexagonal phase formation and lower the bilayer to hexagonal phase transition temperature. The sensitivity of the bilayer to hexagonal phase transition temperature to the presence of additives is at least as great as that which has been observed for any other lipid phase transition.