Miscibility and phase behavior of N-acylethanolamine/diacylphosphatidylethanolamine binary mixtures of matched acyl chainlengths (N=14, 16)

Abstract

The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs), N-myristoylethanolamine (NMEA), and N-palmitoylethanolamine (NPEA), with the corresponding diacyl phosphatidylethanolamines (PEs), dimyristoylphosphatidylethanolamine (DMPE), and dipalmitoylphosphatidylethanolamine (DPPE), respectively, have been investigated by differential scanning calorimetry (DSC), spin-label electron spin resonance (ESR), and (31)P-NMR spectroscopy. Temperature-composition phase diagrams for both NMEA/DMPE and NPEA/DPPE binary systems were established from high sensitivity DSC. The structures of the phases involved were determined by (31)P-NMR spectroscopy. For both systems, complete miscibility in the fluid and gel phases is indicated by DSC and ESR, up to 35 mol % of NMEA in DMPE and 40 mol % of NPEA in DPPE. At higher contents of the NAEs, extensive solid-fluid phase separation and solid-solid immiscibility occur depending on the temperature. Characterization of the structures of the mixtures formed with (31)P-NMR spectroscopy shows that up to 75 mol % of NAE, both DMPE and DPPE form lamellar structures in the gel phase as well as up to at least 65 degrees C in the fluid phase. ESR spectra of phosphatidylcholine spin labeled at the C-5 position in the sn-2 acyl chain present at a probe concentration of 1 mol % exhibit strong spin-spin broadening in the low-temperature region for both systems, suggesting that the acyl chains pack very tightly and exclude the spin label. However, spectra recorded in the fluid phase do not exhibit any spin-spin broadening and indicate complete miscibility of the two components. The miscibility of NAE and diacyl PE of matched chainlengths is significantly less than that found earlier for NPEA and dipalmitoylphosphatidylcholine, an observation that is consistent with the notion that the NAEs are most likely stored as their precursor lipids (N-acyl PEs) and are generated only when the system is subjected to membrane stress.

FIGURE 1

Structures of NMEA and DMPE. The acyl chains in NPEA and DPPE contain 16 C-atoms instead of 14 C-atoms.

FIGURE 2

Calorimetric heating scans of NMEA, DMPE, and their mixtures of different compositions. Samples were dispersed in 10 mM HEPES buffer containing 1 M NaCl and 1 mM EDTA, pH 7.4. Thermograms were recorded at a rate of 10°C/h. The molar composition (NMEA/DMPE) for each sample is indicated.

FIGURE 3

Calorimetric cooling scans of NMEA, DMPE, and their mixtures of different compositions. Samples were dispersed in 10 mM HEPES buffer containing 1 M NaCl and 1 mM EDTA, pH 7.4. Scan rate: 10°C/h. The molar composition (NMEA/DMPE) for each sample is indicated.

FIGURE 4

Binary phase diagrams of (A) NMEA/DMPE and (B) NPEA/DPPE mixtures dispersed in 10 mM HEPES containing 1 M NaCl and 1 mM EDTA, pH 7.4. The phase diagrams have been deduced from the phase boundaries established from DSC endotherms shown in Fig. 2 (for NMEA/DMPE mixture) and Fig. 4 (for NPEA/DPPE mixture). Phase designations are indicated: L_β and L_α are lamellar gel and fluid phases, respectively; is the gel phase of compound C; and S_NMAE and S_NPAE are solid NMAE and NPAE, respectively.

FIGURE 5

Proton-dipolar decoupled 162 MHz ³¹P-NMR spectra of DMPE and NMEA/DMPE binary mixtures of different compositions. Samples were hydrated with 10 mM HEPES containing 1 M NaCl and 1 mM EDTA, pH 7.4. The lipid composition (DMPE:NMEA mol/mol) of the samples and the temperature at which each spectrum was recorded are indicated in the figure. Chemical shifts are referenced to external phosphoric acid.

FIGURE 6

Proton-dipolar decoupled 162 MHz ³¹P-NMR spectra of DPPE and NPEA/DPPE binary mixtures of different compositions. Samples were hydrated with 10 mM HEPES containing 1 M NaCl and 1 mM EDTA, pH 7.4. The lipid composition (DPPE:NPEA mol/mol) of the samples and the temperature at which each spectrum was recorded are indicated in the figure. Chemical shifts are referenced to external phosphoric acid.

FIGURE 7

ESR spectra of 5-PCSL spin label in DMPE, NMEA, and DMPE/NMEA mixtures of the mole ratios indicated. Samples were hydrated with 10 mM HEPES containing 1 M NaCl and 1 mM EDTA, pH 7.4. The temperature at which each spectrum is recorded is indicated in the figure. Total scan width = 100 G.

FIGURE 8

(A) Plots of 2A_max versus mole% of NAE, X_NAE, for the 5-PCSL spin label in various NAE/diacyl PE mixtures. Values of 2A_max for 5-PCSL in mixtures of NMEA and DMPE in the gel phase at 30°C (•) and in the liquid-crystalline phase at 76°C (▪) are shown along with the corresponding values obtained for 5-PCSL in mixtures of NPEA and DPPE in the liquid-crystalline phase at 83°C (Δ). (B) Temperature dependence of 2A_max for 5-PCSL in mixtures of DMPE/NMEA. The composition of the samples is indicated in the figure. See text for details.