Hexagonal phase with ordered acyl chains formed by a short chain asymmetric ceramide
Graphical abstract
Introduction
Sphingolipids comprise a vast family of members composed by a long chain aminediol base N-linked to a fatty acid through an amide bond. The base most usually found is the 18 carbon sphingosine, with a trans double bond at position 4–5, (see Fig. 1), which is itself a bioactive lipid; however other variants with shorter or longer hydrocarbon chains, different hydroxylation level and saturation can be found [1], [2], [3], [4], [5], [6]. In addition, the N-acyl chain can also vary in length (ten in the present case, see Fig. 1), hydroxylation and unsaturation level, although long saturated fatty acyl residues are more often encountered in sphingolipids than in glycerol-based lipids; such variations transduce into largely different molecular packing and phase state [7].
In addition, a great variety of polar groups attached to hydroxyl group at position C1 give rise to the different families of sphingolipids: ceramides, with a single hydroxyl group; sphingomyelins, with phosphorylcholine; cerebrosides, with different sugars; gangliosides, with sugars and sialic acid residues [8]; among others.
The chemical variety of the sphingolipids is responsible for the large structural polymorphism observed due to the intrinsic natural curvature that the polar head group, in relation to the hydrocarbon moiety, imposes to the different lipid families [9]. In this regard, while gangliosides with a complex and charged head group self-assemble as micellar structures of different shapes and size [10], [11], sphingomyelins and cerebrosides form stable bilayers [3]. In the case of ceramides, it has been reported that natural extracts of bovine brain and N-palmitoyl-ceramide can form lamellar stackings [12], a feature also induced by other complex glycosphingolipids and some of their mixtures with phosphatidylcholine [13]. However, when mixed with inverted phase-forming lipids such as phosphatidylethanolamine, it was shown that ceramides N-acylated with fatty acids equal or longer than 8 carbons decreased the temperature of lamellar Lα to hexagonal II transition [14], suggesting that this ceramide stabilized the negative curvature of membranes. On the other hand, when mixed with dipalmitoyl phosphatidylcholine, an increase of the main transition temperature and reduction of bilayer thickness due to chain interdigitation is induced by egg-Ceramide [15].
Ceramides also display the highest melting transition temperatures described in lipid systems [16], [17], [18], owing to tight molecular packing, high Van der Waals interactions and subsequent hydrogen bonding [19]. In bilayer and monolayer membrane model systems, ceramides form highly ordered, solid phases [19], [20], [21] and, when mixed with either glycerophospholipids or sphingolipids, induce phase segregation of condensed domains and complex thermograms [18], [22], [23], [24]. However it should be pointed out that even the condensed C16:0 ceramide can undergo liquid-expanded to condensed transitions depending on temperature, besides other structural transitions in the condensed state [21]. Also, when the N-acyl chain length of ceramides is shortened to less than 14 carbons, expanded phase and expanded to condensed monolayer phase transition at room temperature have also been found in Langmuir monolayers [21].
In the present work, we studied the phase behavior of an asymmetric ceramide N-acylated with the 10 carbons long decanoic acid, C10:0 ceramide, when dispersed in excess water in bulk. Previous studies [21] have shown C10:0 ceramide is able to form stable monolayers with an expanded to condensed transition at 25 mN m−1 at 24 °C. Combining differential scanning calorimetry (DSC), small angle X-ray scattering (SAXS), polarized light microscopy (PLM), wide angle X-ray scattering (WAXS) and Fourier transform infrared spectroscopy (FTIR) we could ascertain the presence of a novel lipid phase below the main transition that consists of an inverted hexagonal phase HII in which the acyl chains of C10:0 ceramide are highly ordered. As far as we know, this is the first report of a curved-tubular (hexagonal) phase with ordered acyl chains in pure lipid systems.
Section snippets
Materials and methods
The lipid N-decanoyl-d-erythro-sphingosine (C10:0 Ceramide, Fig. 1) was from Avanti Polar Lipids (Alabaster, Al, USA) and used without further purification. D2O 99.9% was from Sigma-Aldrich.
C10:0 Ceramide was difficult to hydrate and generally three to four cycles of freeze and thawing should be carried out from −20 °C to 90 °C, followed by extensive bath-sonication. Samples at concentrations higher than 2 mg mL−1 looked opalescent. Unless otherwise indicated, measurements were done in triplicate,
DSC
Ceramide N-acylated with decanoic acid (C10:0 ceramide) has not, to our knowledge, previously been studied by DSC, although thermograms of ceramides with shorter and longer N-acyl chains have been reported [12], [14], [18], [22]. Due to difficulties in preparation of C10:0 ceramide dispersions a quantitative loading of the DSC cell was not reliable, thus values of ΔCp are not reported. Thermograms of C10:0 ceramide showed asymmetric and rather noisy peaks with a weak exotherm at 60 °C and a main
Discussion
In order to rationalize the present results of C10:0 ceramide in bulk, we will first take into account the surface behavior of C10:0 ceramide in planar Langmuir monolayers and compare them with C16:0 sphingomyelin. Previous studies in Langmuir monolayers had demonstrated that C10:0 ceramide at 24 °C showed an expanded to condensed transition [21] at about 25 mN m−1, a surface pressure value higher than the transition pressure of C16:0 sphingomyelin [22], [23], [33], [34] which is about 12 mN m−1.
Conclusions
The asymmetric short chain sphingolipid C10:0 ceramide formed an inverted hexagonal HII phase at full hydration conditions and at temperatures below the main endothermic melting transition. This novel phase with ordered chains is formed as lipid asymmetry reduces the void volumes between the lipid tubules arranged in hexagonal lattice.
Acknowledgements
RGO thanks to the Brazilian Synchrotron Light Laboratory (CNPEM/MCT) for X-ray beamtime at SAXS-2 beamline (projects D11A – SAXS1-10716 and 16019), and at the XRD-1 beamline.
Dr Víctor M Galván Sosa (FaMAF-UNC/CONICET) is acknowledged by his assistance in modelling of the SAXS data and for helpful discussions. The technical assistance of Mr Rafael Gutierrez (Centro Científico Tecnológico-CONICET/Tucumán) for the setting up of infrared spectrometer and experiments is acknowledged.
This work was
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