Spectroscopic evaluation of human tear lipids

Infrared and fluorescence spectroscopies were applied to characterize the molecular conformational/structure and dynamics of human meibum (ML) and tear lipids (SSL). ML lipids contained more CC and CH3 moieties than SSL. SSL contained OH groups that were not apparent in the spectra of ML. The CO stretching band observed in the infrared spectra of SSL and ML revealed that the CO groups are not involved in hydrogen bonds. Bands due to the polar moieties CO and PO2- did not change significantly with increasing temperature, suggesting that they may not play an appreciable thermodynamic role in the lipid hydrocarbon chain phase transition. Components in tears bind to SSL and exclude water at the water-lipid boundary where the polar headgroups of phospholipids are located. If similar interactions occur in vivo at the tear film lipid-aqueous interface, they would reduce the rate of evaporation. The results provide a foundation for future studies to assess possible differences with age and sex in tears from normal and dry eye subjects.     

Concentration dependent cholesteryl-ester and wax-ester structural relationships and meibomian gland dysfunction

BackgroundWith dry eye, the ratio of cholesteryl ester (CE) to wax ester (WE) decreases substantially in meibum, but the functional and structural consequences of this change are speculative. The aim of this study is to confirm this finding and to bridge this gap in knowledge by investigating the effect of varying CE/WE ratios on lipid structure and thermodynamics.MethodsInfrared spectroscopy was use to quantify CE and WE in human meibum and to measure hydrocarbon chain conformation and thermodynamics in a cholesteryl behenate, stearyl stearate model system.ResultsThe CE/WE molar ratio was 36% lower for meibum from donors with dry eye due to meibomian gland dysfunction compared with meibum from donors without dry eye. CE (5 mol %) dramatically increased the phase transition temperature of pure WE from -0.12 °C to 63 °C in the mixture. Above 5 mol % CB, the phase transition temperature increased linearly, from 68.5 °C to 85 °C. In the ordered state, CE caused an increase in lipid order from about 72% trans rotamers to about 86% trans rotamers. Above 10% CE, the hydrocarbon chains were arranged in a monoclinic geometry.ConclusionsThe CE/WE is lower in meibum from donors with dry eye due to meibomian-gland dysfunction. Major conformational changes in the hydrocarbon chains of wax and cholesteryl ester mixtures begin to occur with just 5% CB and above.General significanceCE-WE interactions may be important for in understanding lipid layer structure and functional relationships on the surface of tears, skin and plants.     

Temperature-induced transitions in the structure and interfacial rheology of human meibum

Meibomian lipids are the primary component of the lipid layer of the tear film. Composed primarily of a mixture of lipids, meibum exhibits a range of melt temperatures. Compositional changes that occur with disease may alter the temperature at which meibum melts. Here we explore how the mechanical properties and structure of meibum from healthy subjects depend on temperature. Interfacial films of meibum were highly viscoelastic at 17°C, but as the films were heated to 30°C the surface moduli decreased by more than two orders of magnitude. Brewster angle microscopy revealed the presence of micron-scale inhomogeneities in meibum films at higher temperatures. Crystalline structure was probed by small angle x-ray scattering of bulk meibum, which showed evidence of a majority crystalline structure in all samples with lamellar spacing of 49 Å that melted at 34°C. A minority structure was observed in some samples with d-spacing at 110 Å that persisted up to 40°C. The melting of crystalline phases accompanied by a reduction in interfacial viscosity and elasticity has implications in meibum behavior in the tear film. If the melt temperature of meibum was altered significantly from disease-induced compositional changes, the resultant change in viscosity could alter secretion of lipids from meibomian glands, or tear-film stabilization properties of the lipid layer.     

The interfacial structure of phospholipid bilayers: differential scanning calorimetry and Fourier transform infrared spectroscopic studies of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine and its dialkyl and acyl-alkyl analogs.

The thermotropic phase behavior of aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) and its 1,2-dialkyl, 1-acyl 2-alkyl and 1-alkyl 2-acyl analogs was examined by differential scanning calorimetry, and the organization of these molecules in those hydrated bilayers was studied by Fourier transform infrared spectroscopy. The calorimetric data indicate that substitution of either or both of the acyl chains of DPPC with the corresponding ether-linked hydrocarbon chain results in relatively small increases in the temperature (< 4 degrees C) and enthalpy (< 1 kcal/mol) of the lipid chain-melting phase transition. The spectroscopic data reveal that replacement of one or both of the ester-linked hydrocarbon chains of DPPC with its ether-linked analog causes structural changes in the bilayer assembly, which result in an increase in the polarity of the local environments of the phosphate headgroups and of the ester carbonyl groups at the bilayer polar/apolar interface. The latter observation is unexpected, given that ester linkages are considered to be intrinsically more polar that ether linkages. This finding cannot be satisfactorily rationalized unless the conformation of the glycerol backbones of the analogs containing ether-linked hydrocarbon chains differs significantly from that of diacyl glycerolipids such as DPPC. A comparison of the alpha-methylene scissoring bands and the methylene wagging band progressions of these lipids with the corresponding absorption bands of specifically chain-perdeuterated analogs of DPPC also supports the conclusion that replacement of the ester-linked hydrocarbon chains of DPPC with the corresponding ether-linked analog induces conformational changes in the lipid glycerol backbone. The suggestion that the conformation of glycerol backbones in the alkyl-acyl and dialkyl derivatives of DPPC differs from that of the naturally occurring 1,2-diacyl glycerolipid suggests that mono- and di-alkyl glycerolipids may not be good models of their diacyl analogs. These results, and previously published evidence that DPPC analogs with ether-linked hydrocarbon chains spontaneously form chain-interdigitated gel phases at low temperatures, clearly indicate that the properties of lipid bilayers can be substantially altered by small changes in the chemical structures of their polar/polar interfaces, and highlight the critical role of the interfacial region as a determinant of the structure and organization of lipid assemblies.     

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 °C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C₄₀ ω-ω′ biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to “physiological” conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type α) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type β′). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type α. (3) In all the phases with chains in the α conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 Å. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the α conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains. The possibility is discussed that this phenomenon may reflect an evolutionary response to a challenge of the natural habitat of these archaebacteria.     

Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.     

Molecular arrangements in sphingolipids. Thermotropic phase behaviour of tetracosanoylphytosphingosine

The phase behaviour of a ceramide species containing C₁₈-phytosphingosine and C₂₄-fatty acid was studied by X-ray diffraction methods, in order to elucidate the packing principles of lipids with unequally long hydrocarbon chains. Six solid phases were observed. In five of them, the ceramide molecules have an extended, V-shaped conformation and pack in single layer arrangements with the sphingosine and fatty acid chains forming separate matrices. Differences between these phases are mainly due to thermotropic changes in packing efficiency and thus in tilt of the hydrocarbon chains. The chain packing undergoes transitions from triclinic (T|) to monoclinic (M|) and hexagonal, and between orthorhombic (O⊥) and hexagonal subcell arrangements, respectively. Only one case was observed, in which the molecules pack with their two chains parallelly stacked in a double layer arrangement in which the long fatty acid tails deeply interdigitate between the two opposite layer halves. In a natural membrane containing different lipids, however, long fatty acid tails probably arrange randomly and contribute to the formation of a liquid hydrocarbon zone in the center of the bilayer.     

Behavior of cholesterol and its effect on head group and chain conformations in lipid bilayers: a molecular dynamics study.

Cholesterol molecules were put into a computer-modeled hydrated bilayer of dimyristoyl phosphatidyl choline molecules, and molecular dynamics simulations were run to characterize the effect of this important molecule on membrane structure and dynamics. The effect was judged by observing differences in order parameters, tilt angles, and the fraction of gauche bonds along the hydrocarbon chains between lipids adjacent to cholesterol molecules and comparing them with those further away. It was observed that cholesterol causes an increase in the fraction of trans dihedrals and motional ordering of chains close to the rigid steroid ring system with a decrease in the kink population. The hydrogen-bonding interactions between cholesterol and lipid molecules were determined from radial distribution calculations and showed the cholesterol hydroxyl groups either solvated by water, or forming hydrogen bond contacts with the oxygens of lipid carbonyl and phosphate groups. The dynamics and conformation of the cholesterol molecules were investigated and it was seen that they had a smaller tilt with respect to the bilayer normal than the lipid chains and furthermore that the hydrocarbon tail of the cholesterol was conformationally flexible.     

Investigation into the fluidity of lipopolysaccharide and free lipid A membrane systems by Fourier-transform infrared spectroscopy and differential scanning calorimetry

The phase behaviour, particularly the fluidity within each phase state and the transitions between them, of lipopolysaccharides and of their lipid moiety, free lipid A, of various species of Gram-negative bacteria, especially of Salmonella minnesota and Escherichia coli, has been investigated by applying mainly Fourier-transform infrared spectroscopy and differential scanning calorimetry. For enterobacterial strains, the transition temperatures of the gel----liquid crystalline (beta----alpha) phase transition of the hydrocarbon chains in dependence on the length of the sugar moiety are highest for free lipids A (around 45 degrees C) and lowest for deep rough mutant lipopolysaccharides (around 30 degrees C). Evaluating certain infrared active vibration bands of the hydrocarbon moiety, mainly the symmetric stretching vibration of the methylene groups around 2850 cm-1, it was found that, in the gel state, the acyl chains of lipopolysaccharides and free lipid A have a higher fluidity as compared with saturated and the same fluidity as compared with unsaturated phospholipids. This 'partial fluidization' of lipopolysaccharide below the transition temperature correlates with its reduced enthalpy change at that temperature compared to phospholipids with the same chain length. The fluidity depends strongly on ambient conditions, i.e. on the Mg2+ and H+ content: higher Mg2+ concentrations and low pH values make the acyl chains of free lipid A and lipopolysaccharide preparations significantly more rigid and also partially increase the transition temperature. The influence of Mg2+ is highest for free lipid A and decreases with increasing length of the sugar side chain within the lipopolysaccharide molecules, whereas the effect of a low pH is similar for all preparations. At basic pH, a fluidization of the lipopolysaccharide and lipid A acyl chains and a decrease in transition temperature take place. Free lipid A and all investigated rough mutant lipopolysaccharides exhibit an extremely strong lyotropic behaviour in the beta----alpha melting enthalpy but not in the value of the transition temperature. The phase transition is distinctly expressed only at water concentrations higher than 50-60%. A further increase of the water content still leads to an increase in the phase-transition enthalpy, particularly for lipopolysaccharides with a more complete sugar moiety. The fluidity of the hydrocarbon chains is shown to be an important parameter with respect to the expression of biological activities.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand affinity.

The principal lipid binding protein in tears, tear lipocalin (TL), binds acid and the fluorescent fatty acid analogs, DAUDA and 16-AP at one site TL compete for this binding site. A fluorescent competitive binding assay revealed that apo-TL has a high affinity for phospholipids and stearic acid (Ki) of 1.2 microM and 1.3 microM, respectively, and much less affinity for cholesterol (Ki) of 15.9 of the hydrocarbon chain. TL binds most strongly the least soluble lipids permitting these lipids to exceed their maximum solubility in aqueous solution. These data implicate TL in solubilizing and transporting lipids in the tear film. Phenylalanine, tyrosine and cysteine+ were substituted for TRP 17, the only invariant residue throughout the lipocalin superfamily. Cysteine substitution resulted in some loss os secondary structure, relaxation of aromatic side chain rigidity, decreased binding affinity for DAUDA and destabilization of structure. Mutants of TL, W17Y, and W17F showed a higher binding affinity for DAUDA than wild-type TL. Comparison of the results of the tryptophan 17 substitution in lipocalin with those of tryptophan 19 substitution in beta-lactoglobulin revealed important differences in binding characteristics that reflect the functional heterogeneity within the lipocalin family.     

Conformation of liquid N-alkanes.

The conformations of liquid n-alkanes have been studied using neutron scattering techniques to better understand the conformational forces present in membrane lipid interiors. We have studied hydrocarbon chains having lengths comparable to those found for esterified membrane lipid fatty acids, and find that the steric constraints of packing in the liquid state do not change the conformational distributions of hydrocarbon chains from those imposed by the intrachain forces present in the gas phase. It follows that the central region of membranes containing lipids in the disordered state should contain hydrocarbon chain conformations determined primarily by intrachain forces.     

Studies of mixed-chain diacyl phosphatidylcholines with highly asymmetric acyl chains: a Fourier transform infrared spectroscopic study of interfacial hydration and hydrocarbon chain packing in the mixed interdigitated gel phase.

The mixed interdigitated gel phases of unlabeled, specifically 13C = O-labeled, and specifically chain-perdeuterated samples of 1-O-eicosanoyl, 2-O-lauroyl phosphatidylcholine and 1-O-decanoyl, 2-O-docosanoyl phosphatidylcholine were studied by infrared spectroscopy. Our results suggest that at the liquid-crystalline/gel phase transition temperatures of these lipids, there is a greater redistribution in the populations of free and hydrogen-bonded ester carbonyl groups than is commonly observed with symmetric chain n-saturated diacyl phosphatidylcholines. The formation of the mixed interdigitated gel phase coincides with the appearance of a marked asymmetry in the contours of the C = O stretching band, a process which becomes more pronounced as the temperature is reduced. This asymmetry is ascribed to the emergence of a predominant lipid population consisting of free sn1- and hydrogen-bonded (hydrated) sn2-ester carbonyl groups. This suggests that the region of the mixed interdigitated bilayer polar/apolar interface near to the sn1-ester carbonyl group is less hydrated than is the case with the noninterdigitated gel-phase bilayers formed by normal symmetric chain phosphatidylcholines. In the methylene deformation region of the spectrum, the unlabeled lipids exhibit a pronounced splitting of the CH2 scissoring bands. This splitting is significantly attenuated when the short chains are perdeuterated and collapses completely upon perdeuteration of the long chains, irrespective of whether the long (or short) chains are esterified to the sn1 or sn2 positions of the glycerol backbone. These results are consistent with a global hydrocarbon chain packing motif in which the zigzag planes of the hydrocarbon chains are perpendicular to each other and the sites occupied by long chains are twice as numerous as those occupied by short chains. The experimental support for this chain-packing motif enabled more detailed considerations of the possible ways in which these lipid molecules are assembled in the mixed interdigitated gel phase. Generally, our results are compatible with a previously proposed model in which the mixed interdigitated gel phase is an assembly of repeat units which consists of two phosphatidylcholine molecules forming a triple-chain structure with the long chains traversing the bilayer and with the methyl termini of the shorter chains opposed at the bilayer center. Our data also suggest that the packing format which is most consistent with our results and previously published work is one in which the hydrocarbon chains of each repeat unit are parallel to each other with the repeat units themselves being perpendicularly packed.     

Infrared spectroscopic characterization of the interaction of cationic lipids with plasmid DNA

Fourier transform infrared spectroscopy was used to characterize the interaction of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dioctadecyldimethylammonium bromide with plasmid DNA. The effect of incorporating the neutral colipids cholesterol and dioleoylphosphatidylethanolamine on this interaction was also examined. Additionally, dynamic and phase analysis light scattering were used to monitor the size and zeta potential of the resulting complexes under conditions similar to the Fourier transform infrared measurements. Results suggest that upon interaction of cationic lipids with DNA, the DNA remains in the B form. Distinct changes in the frequency of several infrared bands arising from the DNA bases, however, suggest perturbation of their hydration upon interaction with cationic lipids. A direct interaction of the lipid ammonium headgroup with and dehydration of the DNA phosphate is observed when DNA is complexed with these lipids. Changes in the apolar regions of the lipid bilayer are minimal, whereas the interfacial regions of the membrane show changes in hydration or molecular packing. Incorporation of helper lipids into the cationic membranes results in increased conformational disorder of the apolar region and further dehydration of the interfacial region. Changes in the hydration of the DNA bases were also observed as the molar ratio of helper lipid in the membranes was increased.     

Planar supported bilayer polymers formed from bis-diene lipids by Langmuir-Blodgett deposition and UV irradiation

Substrate-supported lipid bilayers have been prepared from bis-diene functionalized phosphorylcholine (PC) lipids and polymerized by UV irradiation. The overall bilayer structure is largely preserved upon removal from water, although significant loss of material occurs from the upper leaflet of the bilayer, likely due to desorption at the air/water interface. The morphology and surface structure of the bilayer, as observed by AFM, indicate a substantially different arrangement of the lipids in the hydrated and dehydrated states, presumably due to the loss of water from the near surface region. These changes have been correlated with infrared spectral shifts sensitive to the conformation of the hydrocarbon chains. Protein adsorption studies show that rehydrated, polymerized bilayers retain a degree of resistance to BSA adsorption intermediate between model hydrophobic and fluid PC lipid bilayer surfaces. The degree of protein adsorption is correlated with desorption of material from the upper leaflet of the bilayer upon drying, which produces voids at which hydrophobically driven protein adsorption occurs.     

X-ray diffraction and electron microscope study of the interactions of myelin components. The structure of a lamellar phase with a 150 to 180 A repeat distance containing basic proteins and acidic lipids

A variety of phases has been studied: those formed by lipids extracted from myelin, the basic myelin proteins A1 (from the central nervous system) and P1 (from the peripheral nervous system) or other basic proteins. A particularly interesting type of phase was observed which consists of one of the basic proteins of myelin, acidic phospholipids and sulphatides; this phase is lamellar and contains two lipid bilayers in its unit cell. The structure of this phase was determined by the pattern recognition technique and by electron microscope observations of OsO₄-flxed and freeze-etched preparations. It is formed by two different lipid bilayers, one containing mainly the phospholipids with the hydrocarbon chains in a liquid-like conformation and the other containing mainly the sulphatides with at least one fraction of the chains stiff and hexagonally packed. Under the effect of high temperature, or if cholesterol is added, this phase is replaced by other phases which lack the large repeat. The segregation of the lipids and their specific associations with the basic proteins are discussed in relation to the structure of myelin.     

Order-disorder conformational transitions of the hydrocarbon chains of lipids

In many lipid-containing systems (intact membranes, lipid-water and proteinlipid-water phases) the hydrocarbon chains are known to undergo a reversible temperature-dependent transition between a highly disordered (type α) and a partly ordered (type β) conformation; in the β conformation the chains, stiff and all parallel, are packed with rotational disorder according to a two-dimensional hexagonal lattice. This work describes an X-ray diffraction and freeze-fracturing electron microscope study of the phases involved in this conformational transition. Several lipid-water systems were studied: mitochondrial lipids; phosphatidic acid, synthetic lecithin; hen egg lecithin. The conformational transition is found to be a complex phenomenon dependent upon the chemical composition of the lipids, the amount of water and temperature. When the lipid is a pure chemical species the transition involves two phases; one with all the chains in the α conformation the other with all the chains in the β conformation. If the chains are heterogeneous, then from the onset of the transition from type α, they segregate into regions with different conformation, presumably according to their length and degree of saturation. One of the phases (Lαβ) consists of regularly stacked lipid lamellae, each of which is a disordered mosaic of two types of domains; one with the chains in the α, the other in the β conformation. In another phase (Lγ) each lipid lamella is formed by one monolayer of type α and one of type β, joined by their apolar faces. Two other phases (Pγ and Pαβ) display two-dimensional lattices, and consist of lipid lamellae distorted by wave-like ripples, with an ordered segregation of domains in the α and in the β conformation. The number and the structure of the phases involved in the conformational transition are strongly dependent upon the heterogeneity of the hydrocarbon chains and upon the charge and hydration of the polar groups. The results of this study have a bearing on the conformation of the chains in membranes, and on the possible biological significance of conformational transitions.     

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay.

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.     

Thermodynamic and structural characterization of amino acid-linked dialkyl lipids

Using differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR), we determined some thermodynamic and structural parameters for a series of amino acid-linked dialkyl lipids containing a glutamic acid-succinate headgroup and di-alkyl chains: C12, C14, C16 and C18 in CHES buffer, pH 10. Upon heating, DSC shows that the C12, C14 and annealed C16 lipids undergo a single transition which XRD shows is from a lamellar, chain ordered subgel phase to a fluid phase. This single transition splits into two transitions for C18, and FTIR shows that the upper main transition is predominantly the melting of the hydrocarbon chains whereas the lower transition involves changes in the headgroup ordering as well as changes in the lateral packing of the chains. For short incubation times at low temperature, the C16 lipid appears to behave like the C18 lipid, but appropriate annealing at low temperatures indicates that its true equilibrium behavior is like the shorter chain lipids. XRD shows that the C12 lipid readily converts into a highly ordered subgel phase upon cooling and suggests a model with untilted, interdigitated chains and an area of 77.2A(2)/4 chains, with a distorted orthorhombic unit subcell, a=9.0A, b=4.3A and beta=92.7 degrees . As the chain length n increases, subgel formation is slowed, but untilted, interdigitated chains prevail.     

Interaction of a peptide model of a hydrophobic transmembrane alpha-helical segment of a membrane protein with phosphatidylethanolamine bilayers: differential scanning calorimetric and Fourier transform infrared spectroscopic studies.

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic alpha-helical hydrophobic transmembrane peptide, Acetyl-Lys2-Gly-Leu24-Lys2-Ala-Amide, and members of a homologous series of n-saturated diacylphosphatidylethanolamines (PEs). In the lower range of peptide mol fractions, the DSC endotherms exhibited by the lipid/peptide mixtures consist of two components. The temperature and cooperativity of the sharper, higher-temperature component are very similar to those of pure PE bilayers and are almost unaffected by variations in the peptide/lipid ratio. However, the fractional contribution of this component to the total enthalpy change decreases with increases in peptide concentration, and this component completely disappears at higher peptide mol fractions. The other component, which is less cooperative and occurs at a lower temperature, predominates at higher peptide concentrations. These two components of the DSC endotherm can be attributed to the chain-melting phase transitions of peptide-nonassociated and peptide-associated PE molecules, respectively. Although the temperature at which the peptide-associated PE molecules melt is progressively decreased by increases in peptide concentration, the magnitude of this shift is independent of the length of the PE hydrocarbon chain. In addition, the width of the phase transition observed at higher peptide concentrations is also relatively insensitive to PE hydrocarbon chain length, except that peptide gel-phase immiscibility occurs in very short- or very long-chain PE bilayers. Moreover, the enthalpy of the chain-melting transition of the peptide-associated PE does not decrease to 0 even at high peptide concentrations, suggesting that this peptide does not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. The FTIR spectroscopic data indicate that the peptide remains in a predominantly alpha-helical conformation, but that the peptide alpha-helix is subject to small distortions coincident with the changes in hydrophobic thickness that accompany the chain-melting phase transition of the PE bilayer. These data also indicate that the peptide significantly disorders the hydrocarbon chains of adjacent PE molecules in both the gel and liquid-crystalline states relatively independently of lipid hydrocarbon chain length. The relative independence of many aspects of PE-peptide interactions on the hydrophobic thickness of the host bilayer observed in the present study is in marked contrast to the results of our previous study of peptide-phosphatidylcholine (PC) model membranes (Zhang et al. (1992) Biochemistry 31:11579-11588), where strong hydrocarbon chain length-dependent effects were observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Meibum lipid hydrocarbon chain branching and rheology after hematopoietic stem cell transplantation

PurposeMeibum from donors who have had hematological stem cell transplantations (M_HSCT) are susceptible to severe dry eye symptoms and exhibit very high lipid order (stiffness) compared with meibum from donors without dry eye (M_n). Since lipid order could have functional consequences, we compared the rheology and composition of M_n and M_HSCT to measure meibum compositional, structural and functional relationships.MethodsThe rheology and composition was measured using Langmuir trough and ¹H NMR spectroscopy, respectively.ResultsM_HSCT and M_n was studied from 16 to 43 donors, respectively, using NMR spectroscopy. M_HSCT contained significantly 16% more straight chain and 24% less iso-chain hydrocarbons compared with M_n. The cholesteryl ester to wax ester molar ratio, and hydrocarbon chain unsaturation were not significantly different, for M_HSCT compared with M_n.Surface pressure-area isotherms of meibum from 30 donors without dry-eye were grouped into 4 pools (PC) and meibum from 32 donors with dry eye who had hematopoietic stem cell transplantation (PT) were grouped into 3 pools. Above 15 years of age the П_max and (C_s^?1)_max increased with age for both the PC and the PT cohorts. (C_s^?1)_max values were higher for PT samples compared with age matched PC samples, indicating they had higher elasticity and stiffness. A more ordered lipid could contribute to the formation of a discontinuous patchy tear film lipid layer, which in turn results in deteriorated spreading, and decreased surface elasticity.ConclusionsThe composition and rheology of meibum from donors with dry eye and who have had HSCT support the idea from other studies that more ordered meibum may contribute to or be a marker of dry eye.