A deuterium NMR study of labelled n-alkanol anesthetics in a model membrane.

The 2H-NMR spectra of 50 wt.% aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) containing either selectively deuterated 1-decanol (25 mol%) or [2H17]-1-octanol (25 mol%) have been measured as a function of temperature. Both alkanols are potent anesthetics. A detailed carbon-deuterium bond order parameter profile of 1-decanol in liquid crystalline phospholipid dispersions at 50 degrees C was determined from the quadrupolar splittings of 1-decanols deuterated at eight different positions. A maximum order parameter SCD = 0.20 was obtained for [5,5-2H2]-1-decanol, with labels at both ends of the 1-decanol exhibiting reduced order parameters. Explanations for the reduced order towards the hydroxyl group of 1-decanol are discussed in terms of either increased amplitudes of motion or geometric effects due to hydrogen bonding. By comparing the order parameter profile of sn-2 chain deuterated phosphatidylcholine dispersions containing 25 mol% 1-decanol (J.L. Thewalt, S.R. Wassall, H. Gorrissen and R.J. Cushley, Biochim. Biophys. Acta, 817 (1985) 355) with the profile of deuterated 1-decanol in DPPC, we estimate that decanol is approximately parallel to the C-3 to C-13 region of the phosphatidylcholine's sn-2 chain. Variation of the spectral moments M1 with temperature indicates that both 1-decanol and 1-octanol are sensitive to the packing of the lipid in which they are dissolved. Below the phase transition temperature, the 2H-NMR spectra of either 1-decanol (selectively deuterated) or 1-octanol (perdeuterated) are broad powder patterns, characteristic of axially symmetric rotation about the alcohol's long axis. This is in contrast to the 2H-NMR spectra obtained from deuterated phosphatidylcholine under similar conditions, which implies that the phospholipid acyl chain conformations are more restricted than those of the alcohol at these temperatures. From the M1 behavior of the various alkanol chain segments with temperature, the gel to liquid crystalline phase transition is seen to initiate in the middle of the DPPC/1-alkanol bilayer.

Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers.

Aqueous dispersions of egg-phosphatidylcholine and egg-phosphatidylcholine/30 mol% cholesterol containing deuterated tripalmitin or triolein were studied at approx. 25 degrees C by 2H-NMR. Incorporation of tripalmitin into egg-phosphatidylcholine bilayers was found to be less than 0.1 mol%, while the incorporation of triolein is approx. 2.5 mol% in the absence and approx. 0.7 mol% in the presence of cholesterol. The profile of order parameter versus chain position for deuterated triolein suggests that the oleoyl chains of the triacylglycerol have an average orientation such that the C2 chain segments and the segments in the vicinity of the C9-C10 double bond are tilted with respect to the bilayer normal, while all other segments are parallel to the bilayer normal. Longitudinal relaxation times were also determined and indicate that the acyl chains of triolein have a motional behaviour similar to that of phospholipid acyl chains in the bilayer.

Effects of cholesterol on the orientational order of unsaturated lipids in the membranes of acholeplasma laidlawii. A 2H-NMR study.

We have investigated by 2H-NMR the effects of the incorporation of cholesterol on the orientational order of unsaturated lipid acyl chains in the membranes of acholeplasma laidlawii B. This is the only 2H-NMR study to date of the influence of cholesterol in a biological membrane using specifically labelled fatty acids. We observed the characteristic condensing effect of cholesterol on the lipid acyl chain order in the liquid crystalline phase. In terms of the percentage increase in the quadrupolar splittings, the presence of cholesterol has its greatest effect on the methyl end of the labelled oleoyl chains, with a maximum at the C-14 segment. In absolute terms, the perturbation is greatest in the carboxyl end of the chains. The temperature dependence of the 2H spectra for the cholesterol-containing membranes is very similar to that for the cholesterol-free membranes. The broad phase transition of the membrane lipids, which is characteristic for the samples lacking cholesterol, is apparently little affected by the presence of up to 27 mol% cholesterol. In addition, the temperature of onset of the phase transition is not significantly depressed by the presence of cholesterol.

Deuterium magnetic resonance of selectively deuterated cholesteryl esters in dipalmitoyl phosphatidylcholine dispersions.

Dispersions (50 wt% water) containing 95 mol% dipalmitoyl phosphatidylcholine/5 mol% deuterated cholesteryl palmitate (or stearate) were studied using 2H-NMR. Incorporation of ester into the phospholipid bilayer was found to be 0.5 mol% at 50 degrees C. From the profile of 2H quadrupolar splitting vs. chain position, support for an average conformation resembling a 'horseshoe' within the bilayer is obtained. Quadrupolar relaxation times T2e of approx. 250 microseconds and approx. 850 microseconds are measured for cholesteryl palmitate-2,2-d2 and cholesteryl palmitate-16,16,16-d3, respectively, which are less than one-half those obtained for the corresponding positions in dipalmitoyl-d62 phosphatidylcholine. This is ascribed to a slower rate of motion of the ester chain and/or an extra, slow motion of the molecule.

Orientational order of unsaturated lipids in the membranes of Acholeplasma laidlawii as observed by 2H-NMR.

Oleic acid specifically deuterated at fifteen different positions along the chain, including the double bond, was biosynthetically incorporated into the membrane lipids of the microorganism Acholeplasma laidlawii B. A detailed study of the dynamic conformation of these chains was carried out using deuterium nuclear magnetic resonance. The deuterium spectra fourteen different samples were recorded as a function of temperature over the range 0-41 degrees C. Spectra were obtained down to -52 degrees C for the sample enriched with oleic acid deuterated at the C-12' position. Above 20 degrees C, where the lipids are in the liquid crystal phase, a single quadrupolar powder pattern was observed for each C2H2 segment, except for the C-2' position which gave rise to a three-component spectrum characteristic for this position in both model and biological membranes. Simulation of this spectrum indicates that there are two conformations of the lipid molecule in the region of the C-2' segment of the sn-2 chain. The orientationa fluctuations of the fatty acid chain segments in the A. laidlawii membranes are described by the deuterium order parameters, and a striking similarity is shown to exist between the oleate chain conformation of the A. laidlawii membrane and a phospholipid model membrane. Remarkable similarities are also demonstrated in the A. laidlawii membrane enriched in palmitic and oleic fatty acids when the order parameter profiles are plotted at the same reduced temperature. Below 15 degrees C a second component, due to gel phase lipid, starts to appear in the spectra. This broad gel phase component grows at the expense of the liquid crystal phase component as the temperature is reduced. The spectra indicate that the center of the phase transition is at about -12 degrees C, in good agreement with DSC studies.

Deuterium magnetic resonance of selectively deuterated cholesteryl esters in phosphatidylcholine vesicles.

Deuterium nuclear magnetic resonance (2H NMR) experiments have been performed on selectively deuterated cholesteryl palmitate (CP) and cholesteryl stearate (CS) in egg phosphatidylcholine (PC) unilamellar vesicles. Egg PC vesicles were found to incorporate up to 5 mol % ester and to have a mean diameter of 22 nm. Addition of 20 mol % cholesterol decreased the solubility of cholesteryl ester in the bilayer to approximately 2-3 mol %, but the vesicle size remained unchanged. The 2H MNR results reveal that the acyl chains of CP and CS are highly disordered (SCD less than 0.10) both in the presence and in the absence of cholesterol. T1 measurements for selectively deuterated CP and CS in egg PC vesicles indicate that the high degree of disorder of the ester molecule is not associated with an increase in the rate of gauche-trans chain isomerization. Possible explanations for the low order parameters in terms of molecular motions and orientations are offered.

Preparation of specifically dideuterated octadecanoates and oxooctadecanoates.

Sixteen methyl gem dideuterooctadecanoates with two deuterium atoms at positions 2- to 17- and seven oxo esters, 8-oxooctadecanoate-5,5-d(2), 8-oxooctadecanoate-11, 11-d(2), 11-oxooctadecanoate-8,8-d(2), 11-oxooctadecanoate-14,14-d(2), 12-oxooctadecanoate-9,9-d(2), 7-oxooctadecanoate-10,10-d(2) and 13-oxooctadecanoate-16,16-d(2) with two deuteriums on the carbon gamma to the oxo group, have been synthesized. Two prinicipal methods of introducing deuterium were used: preparation of 2,2-dideutero acids by exchange with deuterium oxide followed by chain extension giving dideuteroxooctadecanoates, which were then reduced, as tosylhydrazones, with sodium cyanoborohydride to dideuterooctadecanoates and stepwise introduction by reduction of oxooctadecanoates with sodium borodeuteride, formation of tosylate or mesylate, reduction with lithium aluminum deuteride to tetradeuterooctadecanol and oxidation to dideuterooctadecanoic acid.

Molecular motion and order in single-bilayer vesicles and multilamellar dispersions of egg lecithin and lecithin-cholesterol mixtures. A deuterium nuclear magnetic resonance study of specifically labeled lipids.

Deuterium (2H) nuclear magnetic resonance (NMR) quadrupole splittings and relaxation times have been measured for a variety of specifically deuterated lipids intercalated in lamellar-multibilayer dispersions and single-bilayer vesicles of egg lecithin and lecithin-cholesterol mixtures. The deduced order parameters and relaxation times vary with position of deuteration, acyl chain length, unsaturation, and temperature. The order parameters and spinlattice relaxation times T1 indicate rapid intramolecular motions of restricted amplitude in both the choline head group and hydrocarbon chains. The ordering profile for the acyl chains is similar to that predicted by statistical-mechanical theory. The order parameters yield estimates of the bilayer thickness and linear coefficient of expansion in close agreement with the x-ray determinations. A comparison of the deuterium and electron spin resonance spinprobe order parameters demonstrates the perturbation of the bilayer by the bulky nitroxide probe. The transverse relaxation time T2 for single-bilayer vesicles is quantitatively accounted for by a simple modification of classical relaxation theory which takes into account the modulation of the static quadrupole interaction by rapid local molecular motions and the modulation of the residual quadrupole interaction by the slower overall tumbling of the vesicle. It is unambiguously demonstrated that molecular motion and order in single-bilayer vesicles are very similar to those in lamellar multibilayers. Significant differences occur only for a few segments near the terminal methyl groups of the acyl chains, where the order parameters for vesicles are 10-30% smaller than those found for lamellae. The incorporation of cholesterol in lecithin bilayers is shown to increase the degree of orientational order in vesicles and lamellae, and to increase the hydrodynamic radius of vesicles. Thus, single-bilayer vesicles and multilamellar dispersions of phospholipids are equally useful models for biological membranes. They yield equivalent information about the internal organization and mobility of lipid bilayers, when the spectral manifestations of overall vesicle motion are correctly taken into account.

Leaf wax of oats.

Leaf wax of oats (Kelsey variety) consists of hydrocarbons (5%), esters (10%), free alcohols (45%), free acids (2.5%), ß-diketone (5.5%), hydroxy-ß-diketones (2.5%), and unidentified (29%). Wax on leaf blades contains more free alcohols than wax on leaf sheaths, and wax on the flag leaf sheath contains more ß-diketone than wax on the rest of the plant. Principal hydrocarbons are C29, C31, and C33. The esters, mainly C44-C48 and C52, are probably C18-C22 and C26 esters of hexacosanol. Free alcohols are almost entirely hexacosanol. The ß-diketone is hentriacontane-14, 16-dione. Hydroxy ß-diketones are a mixture of 5-, 6- and 7-hydroxyhentriacontane-14, 16-diones in the proportions 58∶35∶7. The wax also contains a small amount (0.5%) of 1,16-hexacosanediol.