Effect of chain unsaturation on bilayer response to pressure.

Using wide-line deuterium NMR, the effects of pressure on saturated-chain orientational order and gel-to-liquid-crystal phase transition temperature were observed in bilayers of 16:0-18:1 PC-d31 (POPC-d31) and 16:0-18:2 PC-d31 (PLPC-d31). Spectra were recorded for a range of pressures at selected temperatures and for a range of temperatures at selected pressures up to 193 MPa. For 16:0-18:1 PC-d31, the main transition temperature increased by approximately 0.18 K/MPa, a rate that is similar to what is found for bilayers of disaturated PC's. For 16:0-18:2 PC-d31, the increase in transition temperature with pressure was slightly smaller at approximately 0.13 K/MPa. To investigate the isothermal response of chain orientational order to pressure, spectra for each lipid were obtained for three pressures (ambient, 55 MPa, and 110 MPa) near room temperature (approximately 25 degrees C) and for three pressures (ambient, 110 MPa, and 193 MPa) at higher temperature (approximately 40 degrees C). These temperatures were chosen such that the difference between the higher observation temperature and the main transition of 16:0-18:1 PC-d31 would be similar to the difference between the lower observation temperature and the main transition of 16:0-18:2 PC-d31. Application of a given pressure was found to raise the orientational order for all methylene groups on the saturated chain of a particular lipid by roughly similar amounts. For comparable pressure differences, the pressure-induced ordering of the 16:0-18:1 PC-d31 saturated chain at approximately 40 degrees C was greater than that of the corresponding chain in 16:0-18:2 PC- d31 at approximately 25 degrees C. These observations suggest that increasing levels of chain unsaturation may reduce the sensitivity of bilayer order to variations in pressure at corresponding temperatures relative to their ambient pressure transitions.

Heteroacid phosphatidylcholines with different amounts of unsaturation respond differently to cholesterol.

The effectiveness of cholesterol in removing the gel to liquid crystal phase transition of dispersions of pure molecular species of phosphatidylcholines (PC) that is detectable by differential scanning calorimetry (DSC) has been explored. The effect of cholesterol on 16:0-18:0 PC, 16:0-18:1 PC, 16:0-18:2 PC, 16:0-20:4 PC and 16:0-22:6 PC has been determined. Cholesterol caused a concentration-dependent removal of the detectable phase transitions in all cases. It required very little cholesterol to remove the phase transition 16:0-18:2 PC (< 17 mol% of cholesterol in PC). It required > or = 35 mol% cholesterol to remove delta H for 16:0-18:0 PC and 16:0-22:6 PC. About 20-25 mol% cholesterol caused disappearance of the transitional endotherm of 16:0-18:1 PC and 16:0-20:4 PC. The findings indicate that the magnitude of the influence of cholesterol on phospholipid is dependent on the degree of unsaturation in the lipid.

Differential scanning calorimetric studies of aqueous dispersions of phosphatidylcholines containing two polyenoic chains.

The thermotropic behavior of aqueous dispersions of phosphatidylcholines (PC) that contain two identical unsaturated acyl chains, either linoleoyl, alpha-linolenoyl, or arachidonoyl chains, has been investigated by differential scanning calorimetry. These dipolyenoic PC displayed very broad thermal transitions, which occurred over a range of approximately 40 C deg, that started between -77 degrees C (20:4-20:4 PC) and -71 degrees C (18:2-18:2 PC). The enthalpy changes associated with the transitions were small (1 to 2 kcal/mol, 4 to 8 kJ/mol). The broad, low-enthalpy transitions suggest that these lipids might have a very disordered gel phase, or that the transitions from the gel to the liquid crystal might proceed through some intermediate states. The temperatures over which the transitions occurred were influenced only slightly by the number of double bonds between two and four per chain.

Calcium ions and interactions of pulmonary surfactant proteins SP-B and SP-C with phospholipids in spread monolayers at the air/water interface.

Spread monolayers containing hydrophobic pulmonary surfactant protein, SP-B or SP-C, or SP-B/SP-C (2:1, w/w), alone or mixed with dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG), were formed on saline subphases containing calcium ions. Surface pressure-area characteristics of the films of the proteins were not affected by the presence of Ca2+ in the subphase. Calcium ions did not alter the surface properties of the binary and ternary films of DPPC plus either SP-B, or SP-C, or SP-B/SP-C (2:1, w/w). Surface pressure-area isotherms for the spread films of DPPG plus hydrophobic surfactant protein were Ca(2+)-dependent. The exclusion pressures of SP-B, SP-C and SP-B/SP-C (2:1, w/w) from protein-DPPG films in the presence of calcium were lower than the exclusion pressures in the absence of Ca2+. The divalent cation appeared to suppress the ability of SP-C and SP-B/SP-C (2:1, w/w) to remove phospholipid during squeeze-out from their mixed films with DPPG. The effects of Ca2+ on the monolayers of DPPG plus hydrophobic surfactant proteins were consistent with calcium producing diminished lipid-protein interactions, possibly resulting from Ca(2+)-induced changes in the ionization state and molecular packing of DPPG.

Lipid oxidation and gel to liquid-crystalline transition temperatures of synthetic polyunsaturated mixed-acid phosphatidylcholines.

Synthetic preparations of the polyunsaturated phosphatidylcholines 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) and 1-stearoyl-2-alpha-linolenoyl-sn-glycero-3-phosphocholine (SLnPC) were observed to undergo autooxidation sometimes during synthesis and also on storage. Oxidation was also induced by treatment of unoxidized SLPC with ultraviolet irradiation. Oxidation was estimated by thin layer chromatographic, fatty acid and ultraviolet spectral analyses. With limited oxidation, the gel to liquid-crystalline transition temperatures of aqueous dispersions of these lipids were seen to increase. Extensive oxidation led to a reduction in the enthalpies of the transitions. The increases in transition temperatures were consistent with the presence of conjugated double bonds, as shown by increased absorption at 230 nm, and trans double bonds, in the oxidized lipids leading to the creation of more rigid domains within the bilayer. Some of the changes in the transitions, especially the decreasing enthalpy after extensive oxidation, may have occurred because of the presence of small amounts of lysophosphatidylcholine and other oxidation intermediates or breakdown products seen by thin layer analysis. Thermograms of mixtures of unoxidized SLPC with amounts of lysostearoylPC found in the oxidized samples showed, however, that lysoPC likely did not contribute significantly to the increase in transition temperatures. Thin layer analysis suggested that the presence of cross-linked products could have contributed to the observed thermotropic properties.

Temperature dependence of kinetic parameters of (Ca2+ + Mg2+)-ATPase in rabbit and winter flounder sarcoplasmic reticulum.

The effect of temperature on the activity of (Ca2+ + Mg2+)-ATPase in rabbit (R) and winter flounder (F) sarcoplasmic reticulum (SR) has been investigated. The enzymes from the two sources appear to be differently adapted to temperature. (Ca2+ + Mg2+)-ATPase activity of FSR was high at 35 degrees C, and declined to zero at 40 degrees C, whereas RSR was still active above 45 degrees C. Km for ATP increased with temperature in a biphasic fashion for both enzymes. The Km values for FSR were 69-75 microM at lower temperatures (9-18 degrees C) and 80-187 microM at higher temperatures (18-35 degrees C). Values for RSR were 6.5-13 microM below 32 degrees C and 37-186 microM above this temperature. At their respective physiological temperatures the enzyme from both rabbit and flounder exhibited similar Km (70-80 microM). Effective ATP binding enthalpies were 3-5 times lower for FSR than for RSR in both temperature regions. Binding energies increased with temperature 3-4 fold for enzyme in both SR. The enzyme in FSR is suggested to be a more effective catalyst than the one in RSR in the sense that its activation energy for ATP hydrolysis is lower. These variations may arise from dissimilarities in either the protein, or in its surrounding lipid, or both. RSR and FSR are significantly different in the nature of the unsaturated chains in their constituent lipids. The difference in lipid composition might account for some of the deversity in the kinetic parameters.

Cholesterol does not remove the gel-liquid crystalline phase transition of phosphatidylcholines containing two polyenoic acyl chains.

Homoacid (single-acid) phosphatidylcholines containing two linoleate (18:2), arachidonate (20:4), or docosahexaenoate (22:6) chains were dispersed in water to form multilamellar vesicles. The influence of cholesterol on the gel to liquid phase transitions was studied by differential scanning calorimetry (DSC). The pure dipolyenoic phosphatidylcholines exhibited very broad endothermic transitions (widths of 28 to 38 C degrees at scanning rates of 5 C degrees/min) in the temperature range of about -80 degrees C to -30 degrees C. The mixing of cholesterol into the phospholipids in proportions up to 50 mol% had little effect on the temperatures, widths or enthalpy changes associated with the phase transitions. The data suggest that cholesterol does not interfere with the packing of these lipids in the gel state, possibly because the packing is already irregular or because the solubility of cholesterol in these lipids is low, or both.

The influence of unsaturation on the phase transition temperatures of a series of heteroacid phosphatidylcholines containing twenty-carbon chains.

A series of heteroacid sn-1,2 diacyl phosphatidylcholines (PC) with twenty-carbon fatty acyl chains has been synthesized. Each PC contained eicosanoate (20:0) in the sn-1 position and one of a group of eicosaenoic acids with increasing numbers of cis double bonds in the sn-2 position. The double bonds were at positions delta 11 (20:1), delta 11,14 (20:2), delta 11,14,17 (20:3), or delta 5,8,11,14 (20:4). The disaturated PC containing two eicosanoate chains was also studied. Aqueous dispersions of these PC were analyzed by differential scanning calorimetry, and data for the gel to liquid-crystalline transitions (given as PC: Tc (degrees C), Tmax (degrees C), delta H (kcal/mol)) were as follows - 20:0-20:0 PC: 66.8, 68.4, 15; 20:0-20:1 PC: 19.8, 22.2, 8; 20:0-20:2 PC: -4.3, 1.8, 5; 20:0-20:3 PC: 1.2, 4.4, 7; 20:0-20:4 PC: -10.7, -6.8, 3. Double bonds in excess of two per chain did not substantially change the transition temperatures of these heteroacid PC. There was a small effect of the location of the multiple double bonds on the transition temperature. The data is consistent with the model that the transition temperatures are determined by a balance between a decrease in the packing density in the gel and a decrease in the rotational freedom of the chains in the liquid crystal, both caused by the double bonds ((1983) Biochemistry 22, 1466-1473).

Phosphatidylcholine-cholesterol interactions: bilayers of heteroacid lipids containing linoleate lose calorimetric transitions at low cholesterol concentration.

Model membranes composed of cholesterol plus one of two phosphatidylcholines (PC), each containing a saturated and a dienoic acyl chain, have been studied by differential scanning calorimetry. The gel to liquid-crystalline phase transition temperature of 1-palmitoyl-2-linoleoyl PC was -19.5 degrees C and that of 1-stearoyl-2-linoleoyl PC was -13.7 degrees C. The addition of cholesterol to the phosphatidylcholines in aqueous dispersion resulted in the progressive removal of the phase transition as observed by differential scanning calorimetry. Per mole of sterol in the membrane, cholesterol was more effective at reducing the enthalpy change of the phase transitions of these bilayers containing dienoic phosphatidylcholines than it is in eliminating the transition of membranes made with other phospholipids that contain more saturated chains. No transitions in membranes made with palmitoyl-linoleoyl PC or stearoyl-linoleoyl PC could be detected calorimetrically when 17 mol% cholesterol was present.

Epifluorescence microscopic observation of monolayers of dipalmitoylphosphatidylcholine: dependence of domain size on compression rates.

A fluorescence microscopic technique was used to observe phase transitions in monolayers of DPPC. The sizes of the domain structures observed were found to be dependent on the rate of compression of the monolayer. The distribution of domain sizes for different rates of compression were unimodal, but the scatter in the sizes was greater during slow compressions.

Method of purification affects some interfacial properties of pulmonary surfactant proteins B and C and their mixtures with dipalmitoylphosphatidylcholine.

Two methods were employed for preparation of lipid extracts from porcine lung surfactant. Pulmonary surfactant proteins SP-B and SP-C were isolated from the extracts using gel-exclusion chromatography on LH-60 with chloroform:methanol acidified with hydrochloric acid. Monolayers of pure SP-B or SP-C isolated from butanol lipid extracts spread at the air-water interface showed larger molecular areas than those determined in films of SP-B or SP-C isolated from chloroform surfactant extracts. Aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) supplemented with 2.5 and 5.0 wt% of SP-B or SP-C obtained from butanol extracts adsorbed faster to the air-water interface than their counterparts reconstituted with proteins isolated from chloroform extracts. Surface pressure-area characteristics of spread monolayers of DPPC plus SP-B or SP-C did not depend on the method of isolation of the proteins. The diagrams of the mean molecular areas vs. composition for the monolayers of DPPC plus SP-B or SP-C showed positive deviations from the additivity rule, independently of the procedure used for preparation of lipid extract surfactant. Matrix-assisted laser desorption/ionization spectrometry of the proteins isolated from different extraction solvents was consistent with some differences in the chemical compositions of SP-Bs. Butylation of SP-B during extraction of surfactant pellet with butanol may account for the differences observed in the molecular masses of SP-Bs isolated by the two different extraction protocols. The study suggests that the method of purification of SP-B and SP-C may modify their ability to enhance the adsorption rates of DPPC/protein mixtures, and this may be relevant to the formulation of protein-supplemented lipids for exogenous treatment of pulmonary surfactant insufficiency.

Exclusion of fluid lipid during compression of monolayers of mixtures of dipalmitoylphosphatidylcholine with some other phosphatidylcholines.

Monolayers composed of dipalmitoylphosphatidylcholine and one of four fluid phosphatidylcholines have been studied for their ability to attain low minimum surface tension during compression at two different speeds. The minimum surface tension depended on the compression rate and the proportions of the fluid and rigid lipid in the monolayer. The type of fluid lipid used in the monolayer also affected the minimum surface tension.

Palmitoylation of lung surfactant protein SP-C alters surface thermodynamics, but not protein secondary structure or orientation in 1,2-dipalmitoylphosphatidylcholine langmuir films.

Pulmonary surfactant-specific protein, SP-C, isolated from porcine lung lavage, has been deacylated to investigate the role of the two thioester linked palmitoyl chains located near the N-terminus. Surface thermodynamic properties, secondary structure, and orientation of native and deacylated SP-C in 1, 2-dipalmitoylphosphatidylcholine (DPPC) monolayers has been characterized by combined surface pressure-molecular area (pi-A) isotherms and infrared reflection-absorption spectroscopy (IRRAS) measurements. The isotherms indicate that deacylation of SP-C produces more fluid monolayers at pressures less than 30 mN m-1. The helical secondary structure and tilt angle (70-80 degrees relative to the surface normal) of SP-C remained essentially unchanged upon deacylation in DPPC monolayers at a surface pressure approximately 30 mN m-1. The results are consistent with a model that acylation of SP-C may influence the rapid protein-aided spreading of a surface-associated surfactant reservoir, but not the structure of DPPC or SP-C in the monolayer at higher surface pressures.

The effect of changes in gramicidin conformation on bilayer lipid properties.

The effects of two different gramicidin conformations on lipid phase behaviour and dynamics are compared. Samples of chain-perdeuterated dimyristoylphosphatidylcholine containing gramicidin were first prepared with gramicidin in a state having a circular dichroism spectrum generally identified as corresponding to the non-channel conformation. The effects, on bilayer lipid properties, of gramicidin in this conformation were then determined using deuterium nuclear magnetic resonance measurements of acyl chain orientational order and transverse relaxation times as a function of temperature. These samples were then incubated at 65 degrees C to convert the gramicidin to a state with a circular dichroism spectrum of the type generally identified with the channel conformation. The nuclear magnetic resonance measurements were then repeated. In the gel phase, it was found that transverse relaxation time and chain orientational order of the lipid were insensitive to gramicidin conformation. In the liquid crystalline phase, gramicidin in the channel conformation was found to have a slightly larger effect on transverse relaxation and orientational order than gramicidin in the non-channel conformation. The perturbation of the phase behavior by gramicidin was found to be relatively insensitive to gramicidin conformation.

Intestinal absorption and lymphatic transport of fish oil (MaxEPA) in the rat.

Adult male, chow-fed Sprague-Dawley rats were given intraduodenally a bolus of emulsion of 0.5 ml of fish oil (MaxEPA) or olive oil plus 0.5 ml of 20 mM sodium taurocholate. Intestinal lymph was collected from a cannula in the main intestinal lymph trunk for various times after oil administration. There was no difference in the absorption of either type of oil over 6 and 24 h, over which times about 40 and 70% of the administered dose was taken up. For MaxEPA, the flux of triacylglycerols remained at a basal level of 0.07 mumol/min for 30 min, after which it rose rapidly to a maximum of 0.87 mumol/min between 90 and 120 min. The flux was 0.4 mumol/min for the subsequent 4 h. After 30 min, the composition of the lymph triacylglycerols began to change to show the presence of large proportions of fatty acyl chains that were characteristic of fish oil, especially eicosapentaenoate (20:5(n-3] and docosahexaenoate (22:6(n-3]. The composition of the lymph remained fairly similar to that of the fish oil for up to 6 h, the last time point at which detailed analysis was done. The docosahexaenoate in the triacylglycerols of the fish oil was primarily in the sn-2 position of glycerol, whereas a more random distribution of eicosapentaenoate over all glycerol positions was found. The positional distribution of the acids in the lymph triacylglycerols was similar to that in the fish oil. There was no evidence of substantial chain elongation or shortening during absorption. The results indicate that fish oil is effectively absorbed from the rat intestine without substantial alteration in the acyl chains of the triacylglycerols.

Intestinal absorption of fish oil in rats previously adapted to diets containing fish oil or corn oil.

A study was conducted to evaluate whether the composition of previous dietary fat affects the absorption and composition of lymph obtained after a meal of fish oil. Adult male Sprague-Dawley rats were fed diets containing either corn oil or fish oil (MaxEPA) for 2 weeks. They were then given intraduodenally a bolus of an emulsion of 0.5 ml of fish oil plus 0.5 ml of 20 mM sodium taurocholate. Intestinal lymph was collected from a cannula in the main intestinal lymph trunk for various times after oil administration. Rats proportion of the test dose fo fish oil than those fed corn oil. There was an effect of previous diet on the fatty acid composition of the lymph. Rats fed fish oil had a higher percentage of eicosapentaenoic and docosahexaenoic acids in the lymph lipids than those fed corn oil while those fed corn oil had a higher percentage of linoleic acid. These results rule out decreased intestinal absorption as a mechanism for the hypotriacylglycerolemic effect of dietary fish oils. They also indicate a significant contribution of endogenous lipids to the fatty acids in lymph.

Sarcoplasmic reticulum from rabbit and winter flounder: temperature-dependence of protein conformation and lipid motion.

A comparative study of lipids and proteins in sarcoplasmic reticulum (SR) from rabbit and flounder has been undertaken. The protein/phospholipid ratio (w/w) was 3:1 in flounder SR (FSR) and 2.2:1 in rabbit SR (RSR). Both membranes had similar contents of PC (70%) and PI (6%). PE constituted 15% in RSR and 21% in FSR. PS and sphingomyelin were minor components of both SR (less than 4%). There were differences in the unsaturated chains of the total lipid extracts, PC, PE, and PI between FSR and RSR. RSR was high in linoleate and arachidonate while FSR contained substantial amounts of eicosapentaenoate and docosahexaenoate. FTIR spectroscopy revealed that the lipids of both membranes did not undergo a phase transition between 0 and 50 degrees C. The lipids were in the liquid-crystalline state at physiological temperatures and underwent monotonic increases in conformational disorder as the temperature was raised. CD spectra indicated higher content of alpha-helical structure of proteins in RSR than in FSR. Increasing temperature caused diminution of alpha-helix content. Relatively large decreases in ellipticity were observed between 20 degrees C and 40 degrees C for FSR and 30 degrees C and 60 degrees C for RSR. Measurements of intrinsic tryptophan fluorescence as a function of temperature gave similar results for membrane proteins in both FSR and RSR. The rate of change of tryptophan fluorescence and fluorescence lifetimes was constant over the temperature ranges studied, and no abrupt shifts in fluorescence occurred in the temperature regions where ellipticity decreased rapidly.

Characterization of bovine surfactant for infants with respiratory distress syndrome.

Exogenous surfactant treatment of surfactant-deficient disease states is now under study in a number of centers, using a variety of surfactant preparations. We have chosen one preparation because of its current and potential clinical usefulness, and we have characterized it using selected tests and assays that we thought would be necessary (although not necessarily sufficient) to justify extended clinical use. We found its lipid composition to resemble that of other surfactants derived from lung mince. There is little variation among several batches with regard to lipid composition or surface tension-lowering capability. Morphologic heterogeneity occurs in individual samples of pelleted material studied by electron microscopy. Arterial oxygenation is improved when the material is administered to animals depleted of surfactant. A low molecular weight protein was identified that reacted with antibody that specifically binds nonserum surfactant proteins in a number of animal species (including human and cow). The characteristics of this surfactant preparation should be useful for comparison as newer and simpler products become available.

Inhibition of pulmonary surfactant by plasma from normal adults and from patients having cardiopulmonary bypass.

Plasma from normal adults and from children and adults having cardiopulmonary bypass inhibited the ability of pulmonary surfactant to reach low surface tension in proportion to the amount of protein added. No increase in the extent of inhibition of surfactant action per milligram of protein was seen in plasma taken before or after bypass in adults or children.

Surface respreading after collapse of monolayers containing major lipids of pulmonary surfactant.

Isotherms have been obtained near 37 degrees C for a series of repetitive compressions and expansions of monolayers that contain major components of lung surfactant. The minimum surface tension or maximum surface pressure which could be achieved under conditions of dynamic compression, and the rate of return of lipid from excluded phase to the monolayers were measured. Monolayers of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or of DPPC plus 10 or 30 mol% of the calcium salt of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG) (POPG-Ca) achieved very high surface pressures or low surface tensions (near 0 mN m-1), but they showed no return of material from the collapse phases under the test conditions. Monolayers of POPG-Ca alone collapsed at relatively low surface pressures (high surface tensions), but showed good return of material from the collapse phase into the monolayer. Monolayers containing more complex mixtures of lipids (DPPC, phosphatidylglycerol (PG), unsaturated phosphatidylcholine (PC), cholesterol (chol] in ratios similar to those found in surfactant achieved minimum surface tensions intermediate between those of monolayers with less complex compositions. These more complex mixtures showed a better rate of return of lipids from the collapse phases to the monolayer than did simple DPPC-POPG mixtures. 31P-NMR and differential scanning calorimetric investigations of the mixture DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POP G/DPPG/chol (10:4:2:1:3) showed that in the bulk phase at 37 degrees C, it was in bilayers in the liquid-crystalline state.