Direct imaging of domains in the Lbeta' state of 1,2-dipalmitoylphosphatidylcholine bilayers.

A near-field scanning optical microscope was used to study domain formation and evolution in single-component supported lipid bilayers in the gel (L(beta') state. Results on 1,2-dipalmitoylphosphatidylcholine (DPPC) bilayers on glass substrates at room temperature are presented. The domain structure is determined by means of the optical anisotropy of the sample, which arises because DPPC molecules are tilted at theta approximately 32 degrees with respect to the bilayer normal [J. F. Nagle and S. Tristram-Nagle, Biochim. Biophys. Acta 1469, 159 (2000)]. From the measurements we obtain the difference in the index of refraction for the directions parallel and perpendicular to the acyl chains of the lipid molecules, Delta(n)=0.37+/-0.12, in good agreement with calculated and measured values. Direct evidence of the existence of domains in the L(beta') state is provided. These domains, defined as the correlation of the tilt angle theta, are found to be 1-2 microm across. Furthermore, it was found that they are robust under single-lipid-molecule diffusion, remaining unchanged over periods of hundreds of minutes.

Solid-state NMR structure determination of melittin in a lipid environment.

Solid-state (13)C NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-(13)C]Gly3-[2-(13)C]Ala4, [1-(13)C]Gly3-[2-(13)C]Leu6, [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-(13)C]Gly3-[2-(13)C]Ala4 and [1-(13)C]Gly3-[2-(13)C]Leu6 were consistent with alpha-helical structure in the N-terminus irrespective of environment. The internuclear distances measured in [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees ) than in lyophilized powder (121 degrees -139 degrees ) or crystals (129 degrees ). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (approximately 160 degrees ). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.

Formation of inverted hexagonal phase in SDPE as observed by solid-state (31)P NMR.

Docosahexaenoic acid (DHA), the longest and most unsaturated fatty acid commonly found in biological membranes, is known to affect various membrane properties. In a variety of cell membranes, DHA is primarily incorporated in phosphatidylethanolamines, where its function remains poorly understood. In order to understand the role of DHA in influencing membrane structure, we utilize (31)P NMR spectroscopy to study the phase behavior of 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanolamine (SDPE) in comparison to 1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine (POPE) from 20 to 50 degrees C. Spectra of SDPE phospholipids show the formation of inverted hexagonal phase (H(II)) from 20 to 50 degrees C; in contrast, POPE mutilamellar dispersions exist in a lamellar liquid-crystalline phase (L(alpha)) at the same temperatures. The ability of SDPE to adopt nonbilayer phases at a physiological temperature may indicate its role in imparting negative curvature stress upon the membrane and may affect local molecular organization including the formation of lipid microdomains within biological membranes.

Molecular organization of cholesterol in polyunsaturated phospholipid membranes: a solid state 2H NMR investigation.

We compared the molecular organization of equimolar [3alpha-2H1]cholesterol in 18:0-18:1PC (1-stearoyl-2-oleoylphosphatidylcholine), 18:0-22:6PC (1-stearoyl-2-docosahexaenoylphosphatidylcholine), 18:0-20:4PC (1-stearoyl-2-arachidonylphosphatidylcholine) and 20:4-20:4PC (1,2-diarachidonylphosphatidylcholine) bilayers by solid state 2H NMR. Essentially identical quadrupolar splittings (delta v(r) = 45 +/- 1 kHz) corresponding to the same molecular orientation characterized by tilt angle alpha0 = 16 +/- 1 degrees were measured in 18:0-18:1PC, 18:0-22:6PC and 18:0-20:4PC. A profound difference in molecular interaction with dipolyunsaturated 20:4-20:4PC, in contrast, is indicated for the sterol. Specifically, the tilt angle alpha0 = 22 +/- 1 degrees (derived from delta v(r) = 37 +/- 1 kHz) is greater and its membrane intercalation is only 15 mol%.

Rapid deconvolution of NMR powder spectra by weighted fast Fourier transformation.

The resolution enhancement conferred by numerical deconvolution of powder pattern spectra to spectra characteristic of a single alignment greatly simplifies solid state NMR spectral analysis. This is especially beneficial when the spectrum is a superposition of signals from multiple environments or sites of labelling. We have developed an innovative method to deconvolute (depake) spectra governed by axially symmetric second rank tensor interactions which possess a P2(cos theta) dependence upon orientation, where theta is the angle between the symmetry axis and the external magnetic field. Our approach differs substantially from previously published procedures which are iterative or require matrix inversion and, hence, are slow. The new method, instead, utilizes weighting functions in time and frequency domains to facilitate a rapidly executed solution based upon fast Fourier transformation (FFT). Its efficacy is demonstrated with 2H and 31P NMR data for model membranes.

Pulsed field gradient-spin echo NMR studies of water diffusion in a phospholipid model membrane.

Water diffusion in the lamellar phase of egg phosphatidylcholine (egg PC)-water was studied by 1H NMR using the pulsed field gradient-spin echo method. The curvature of diffusion plots obtained with egg PC-water mixtures indicates that water diffusion is highly anisotropic with respect to lipid lamellae. This was confirmed by measurements made on macroscopically aligned egg PC-water as a function of orientation that categorically establish DII/DI >> .1, where the respective subscripts refer to parallel and perpendicular to the lipid bilayer. A smooth, monotonic dependence on water concentration was observed for water diffusion in aligned egg PC-water, varying at 25 degrees C from DII = 1.2 x 10(-10) m2 s 1 at n = 4.9 mol water/lipid to DII = 4.0 x 10(-10) m2 s-1 at n = 18.6 mol water/lipid. The diffusion is approximately a factor of 10 slower than in pure water because of water binding and restriction to translational motion within the aqueous layer. No evidence for a sudden drop in water diffusion coefficient at a specific water content, as previously reported with egg PC-water mixtures (Lange and Gary Bobo. 1974. J. Gen. Physiol 63:690-706), was detected. A morphological reorganization of lamellar domains, which in random orientational distribution comprise lipid-water mixtures, is the likely explanation. The study of aligned lipid-water systems is manifestly preferable.

Cholesterol condensation of alpha-linolenic and gamma-linolenic acid-containing phosphatidylcholine monolayers and bilayers.

Cholesterol is demonstrated to condense phosphatidylcholine (PC) monolayers and bilayers containing stearic acid in the sn-1 position and alpha-linolenic acid in the sn-2 position (18:0, alpha-18:3 PC) but has no effect when gamma-linolenic acid occupies the sn-2 position (18:0,gamma-18:3 PC). Cholesterol-induced condensation is measured by area/molecule determinations made on monolayers using a Langmuir trough, while condensation in bilayers is followed by the fluorescent dyes merocyanine (MC540) and dansyllysine. Permeability to erythritol is also demonstrated to be diminished by cholesterol for the condensable 18:0,alpha-18:3 PC bilayer membranes but not the 18:0,gamma-18:3 PC membranes. alpha- and gamma-linolenic acid are isomers containing 18 carbons and three unsaturations. Both fatty acids have unsaturations at positions 9 and 12 and differ only in the location of the third unsaturation, at either position 6 for gamma-linolenic acid (an omega-6 fatty acid) and at position 15 for alpha-linolenic acid (an omega-3 fatty acid). Here lipid-cholesterol interaction is used to distinguish the effect of position of unsaturation on membrane structure.

2H NMR studies of isomeric omega 3 and omega 6 polyunsaturated phospholipid membranes.

The properties of aqueous multilamellar dispersions of [2H31]16:0-alpha 18:3 PC (1-[2H31]palmitoyl-2-cis,cis,cis-octa-9,12,15- trienoylphosphatidylcholine) and of [2H31]16:0-gamma 18:3 PC (1-[2H31]palmitoyl-2-cis,cis,cis-octa-6,9,12-trienoylphosphatid ylcholine) were compared by broadline 2H NMR spectroscopy. These isomeric phospholipids differ only in the location of the unsaturations in the sn-2 chain. The alpha 18:3 chain has double bonds at delta 9, 12, and 15 positions whereas in the gamma 18:3 chain they are at positions delta 6, 9, and 12. Moment analysis of spectra recorded as a function of temperature reveals dramatically distinct phase behavior for the two isomers. The gel to liquid crystalline transition for [2H31]16:0-alpha 18:3 PC membranes exhibits broad hysteresis which is characterized by a mid point temperature of -9 degrees C and -20 degrees C on heating and cooling, respectively. In contrast, the phase transition of [2H31]16:0-gamma 18:3 PC membranes does not exhibit hysteresis and occurs over a lower temperature range centred on -27 degrees C. Appreciably different molecular ordering also exists within the membranes in the liquid crystalline state. Average order parameters SCD are smaller in [2H31] 16:0-alpha 18:3 PC than in [2H31]16:0-gamma 18:3 PC by 10% at the same temperature and by 20% at equal reduced temperature. Smoothed order parameter profiles generated from depaked spectra clarify the nature of the difference.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and dynamic membrane properties of alpha-tocopherol and alpha-tocotrienol: implication to the molecular mechanism of their antioxidant potency.

d-alpha-Tocopherol and d-alpha-tocotrienol are two vitamin E constituents having the same aromatic chromanol "head" but different hydrocarbon "tails". alpha-Tocotrienol has been shown to be more potent in protecting against free radical-induced oxidative stress than alpha-tocopherol. Simple models of phospholipid membrane systems were used to investigate the mechanism of the antioxidant potency of alpha-tocotrienol in terms of its effects on membrane order and reorientation dynamics. Chemiluminescence and fluorescence measurements demonstrated that alpha-tocotrienol exhibits significantly greater peroxyl radical scavenging potency than alpha-tocopherol in phosphatidylcholine liposomes, whereas both antioxidants have identical activity in hexane. This suggests that the antioxidant potency of alpha-tocotrienol requires the membrane environment. When alpha-tocopherol and alpha-tocotrienol were examined for their effects on phospholipid molecular order using conventional ESR spin labeling with 5- and 16-position-labeled doxylstearic acid, although both vitamin E constituents disordered the gel phase and stabilized the liquid-crystalline phase, no differences were observed between the effects of the two compounds. A slightly greater increase (19% vs 15%) in ordering of the liquid-crystalline state due to alpha-tocopherol, however, was discerned in noninvasive 2H NMR experiments. The difference is most noticeable near C10-C13 positions of the phospholipid chain, possibly suggesting alpha-tocotrienol is located closer to the membrane surface. Saturation-transfer ESR, furthermore, revealed that on the time scale tau c = 10(-7)-10(-3) s the rates of rotation about the long molecular axis and of the wobbling motion of the axis are modified to differing extents by the two forms of the vitamin E.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of nutrient intake by deuterium dilution in premature infants.

To assess whether a simple nonrestrictive method of determining nutrient intake could be applied to premature infants, we compared actual measured formula intake during a 7-day period with intake calculated from deuterium dilution in 13 hospitalized, growing, premature newborn infants. An oral dose of deuterium oxide (D2O) was administered, and urine samples were analyzed by deuterium nuclear magnetic resonance spectrometry for D2O concentration. Using an exponential model, we calculated formula intake from the decline in D2O concentration during the 7-day study period. Intake as assessed by the deuterium model correlated well with actual intake (r = 0.93; p < 0.001). However, because the deuterium dilution model measures both dietary and nondietary water intake (metabolic and cutaneous water influx), deuterium dilution-derived intake exceeded actual intake by 25 +/- 18 ml/kg per day (16% +/- 11%). When corrections were applied to account for nondietary water intake, deuterium dilution-derived nutrient intake (160 +/- 30 ml/kg per day) closely approximated actual intake (155 +/- 17 ml/kg per day). If corrections are made for nondietary water intake, the deuterium dilution method may be a useful nonrestrictive method of measuring nutrient intake in a variety of neonatal populations.

Use of merocyanine (MC540) in quantifying lipid domains and packing in phospholipid vesicles and tumor cells.

The fluorescent probe merocyanine (MC540) reports qualitatively on several membrane events. Here we demonstrate that MC540 fluorescence can quantify the degree of coexisting liquid-crystalline and gel states in mixed monotectic phosphatidylcholine (PC) bilayers. The probe exhibits disparate fluorescence wavelength maximas and and intensities when incorporated into liquid-crystalline and gel state membranes. The fluorescence measurements partitioning of the EPR spin probe TEMPO between the aqueous environment and the membrane fluid phase. While both techniques can accurately assess the phase transition of synthetic PCs, only MC540 can distinguish between liquid-crystalline phases of different composition. MC540 fluorescence for single-component PC bilayers correlates quantitatively with estimates of the area/molecule determined from surface area/pressure isotherms of lipid monolayers, whereas partitioning of TEMPO fails to assess the relative degree of lipid packing in various fluid state membranes. Additionally, MC540 fluorescence characterizes the interaction of cholesterol with membranes made from condensable (18:0, 18:1-PC) and non-condensable (18:0, 22:6-PC) lipids. Finally MC540 distinguishes tumor cell membranes differing only in the amount of docosahexaenoic acid (DHA). Thus we conclude that MC540 can be used quantitatively to study phospholipid packing and membrane phases with lipid vesicles and to sense subtle differences in the arrangement of phospholipids in biological membranes.

Effects of dietary fish oil on plasma high density lipoprotein. Electron spin resonance and fluorescence polarization studies of lipid ordering and dynamics.

Dietary fish oils are implicated in reducing the incidence of coronary heart disease, perhaps by altering the properties of plasma lipoproteins. The hypothesis that omega-3 polyunsaturated fatty acids (PUFA) in fish oils produce changes in lipid ordering and dynamics within high density lipoprotein (HDL), thereby potentially modifying cholesterol transport, is investigated here. Rabbits were fed a diet supplemented with either 10% (by weight) menhaden oil (MO), a fish oil rich in omega-3 PUFAs, or hydrogenated cottonseed oil for a period of 12 weeks. HDL was isolated by sequential flotation ultracentrifugation from plasma drawn every 2 weeks. Gas chromatography confirmed that the predominant omega-3 PUFAs of fish oils, eicosapentaenoic 20:5 and docosahexaenoic 22:6 acids, were only incorporated into the triglyceride, phospholipid, and cholesteryl ester constituents of lipoproteins from rabbits on the MO diet. ESR of 5- and 16-doxyl stearic acids demonstrates that molecular order and dynamics within the outer monolayer of HDL is virtually unaffected. In contrast, ESR of cholesteryl 12-doxyl stearate indicates order is less within the inner apolar core of the lipoprotein for the MO diet than for the hydrogenated cottonseed oil diet. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene supports this finding. The greater disorder detected within HDL from rabbits fed fish oil may result in an enhancement of cholesterol exchange between lipoproteins and between lipoproteins and cells, which may have anti-atherogenic ramifications.

Interaction of alpha-tocopherol with fatty acids in membranes and ethanol.

The techniques of fluorescence polarization, ultraviolet light absorbance and fluorescence quenching by acrylamide are used to probe the structural role of alpha-tocopherol in phospholipid bilayers. Using 1,6-diphenyl-1,3,5-hexatriene (DPH) and a series of (anthroyloxy)stearic acid (AS) fluorescence probes, alpha-tocopherol is shown to increase fluidity and decrease order of gel state bilayers, and to decrease fluidity and increase order of bilayers in the liquid crystalline state. More complex behavior is noted for bilayers made from mixed acyl chain phosphatidylcholines (PCs) where the sn-1 position is saturated and the sn-2 position unsaturated compared to bilayers composed of PCs where both acyl chains are either saturated or unsaturated. Complexation between alpha-tocopherol and either free fatty acids or fatty acids esterified to the sn-2 position of PCs is indicated by ultraviolet light absorbance in both organic solution and in lipid bilayers. The strength of the complexes, expressed as interaction constants, are dependent upon the number of acyl chain unsaturations from 0 (stearic acid), to 6 (docosahexaenoic acid). Relation of the strength of these complexes to the degree of acyl chain unsaturation is confirmed by monitoring the fatty acid protection from acrylamide bleaching of alpha-tocopherol. These experiments suggest that the extent of acrylamide bleaching is related to the extent of association with the fatty acids.

Electron spin resonance study of the interaction of alpha-tocopherol with phospholipid model membranes.

The effect of up to 20 mol% incorporation of alpha-tocopherol on acyl chain order and dynamics in liquid crystalline phosphatidylcholine (PC) membranes was studied as a function of acyl chain unsaturation by electron spin resonance (ESR) of 5-, 7-, 12- and 16-doxyl spin labelled stearic acids intercalated into the membrane. Order parameters S in the upper portion of the chain (positions 5 and 7) and correlation times tau C in the lower portion (positions 12 and 16) determined from the ESR spectra indicate that in general alpha-tocopherol restricts acyl chain motion within the membrane. The magnitude of the increases in order appears to be dependent upon phospholipid molecular area, being the greatest (up to 15%) in saturated dimyristoylphosphatidylcholine (14:0-14:0 PC) which possesses a relatively small area per molecule as opposed to much smaller increases (less than 3%) in unsaturated PC membranes of larger molecular area. This behavior is interpreted as incompatible with the hypothesis of Lucy and coworkers (A.T. Diplock and J.A. Lucy (1973) FEBS Lett. 29, 205-210), who proposed that membranes are structurally stabilized by interactions between the phytyl side chain of alpha-tocopherol and the polyunsaturated chains of phospholipids.

Plant sterol inhibition of abscisic acid-induced perturbations in phospholipid bilayers.

Abscisic acid (ABA)-induced phospholipid bilayer perturbations (permeability and lipid vesicle aggregation) are shown to be reversed by incorporation of a commercially available mixture of plant sterols (60% beta-sitosterol, 27% campesterol and 13% dihydrobrassicasterol) into the membranes. As little and 5 membrane mol% plant sterol inhibits ABA-stimulated permeability of both saturated and unsaturated mixed phosphatidylcholine/phosphatidylethanolamine bilayers to the fluorescent anion carboxyfluorescein by more than 50%. The same conclusion was reached by an osmotic swelling technique for the uncharged permeant solute erythritol. Hormone-induced carboxyfluorescein permeability to mixed acyl chain phosphatidylcholine bilayers was similarly inhibited by the sterols, but only if the membranes were tested at a temperature where liquid crystal and gel states coexist. The plant sterols were also shown to prevent the ABA-induced fusion of mixed phosphatidylcholine/phosphatidylethanolamine bilayers. The ABA effect on membranes is inhibited equally by plant sterols as well as cholesterol. From these experiments a possible role is suggested for plant sterols in controlling the mode of action of ABA.

A comparison of the effects of linolenic (18:3 omega 3) and docosahexaenoic (22:6 omega 3) acids on phospholipid bilayers.

The class of long chain polyunsaturated fatty acids known as omega-3 are believed to be involved in prevention of a number of human afflictions. The mode of action for two of the most common omega-3 fatty acids, linolenic 18:3 delta 9,12,15 and docosahexaenoic 22:6 delta 4,7,10,13,16,19 (DHA), is not known. One suggestion is that they may be incorporated into membranes and there provide some specific function. Here we compare the effects of DHA and its metabolic precursor linolenic acid on the membrane properties of fluidity, fusion and permeability. The fatty acids were investigated as both free fatty acids and mixed chain 18:0, 18:3 and 18:0, 22:6 phosphatidylcholines (PCs). Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and a series of anthracene stearic acid probes indicates 20 mol% incorporation of either fatty acid into dipalmitoylphosphatidylcholine bilayers broadens and depresses the temperature of the phase transition, but has almost no effect on fluidity in the liquid crystalline state. Similar fluidity was also observed in the liquid crystalline bilayers of the mixed chain PCs using the same set of fluorescent fatty acid probes. In contrast, DHA as a free fatty acid or as part of a mixed chain PC, causes a much greater enhancement than linolenic acid of the rates of fusion and permeability as monitored by fluorescence resonance energy transfer and aqueous compartment mixing (fusion) and by lipid vesicle swelling in isotonic erythritol, (permeability). These experiments establish a clear distinction between the effects of linolenic acid and DHA in membranes.

Abscisic acid enhances aggregation and fusion of phospholipid vesicles.

The plant hormone abscisic acid (ABA) is shown to enhance the aggregation and fusion of small unilamellar lipid vesicles composed of 80 mol% dimyristoylphosphatidylcholine (DMPC) and 20 mol% dimyristoylphosphatidylcholine (DMPE). Aggregation and fusion did not occur with single component (100 mol%) DMPC vesicles. Fusion was followed by two fundamentally different techniques, fluorescence resonance energy transfer which monitors intermixing of bilayers and ANTS-DPX which monitors intermixing of the sequestered aqueous interiors. It is suggested that a previously unreported role of ABA may be as a membrane fusagen.