Cholesterol strongly affects the organization of lipid monolayers studied as models of the milk fat globule membrane: Condensing effect and change in the lipid domain morphology

Abstract

The biological membrane that surrounds the milk fat globules exhibits phase separation of polar lipids that is poorly known. The objective of this study was to investigate the role played by cholesterol in the organization of monolayers prepared as models of the milk fat globule membrane (MFGM). Differential scanning calorimetry and X-ray diffraction experiments allowed characterization of the gel to liquid crystalline phase transition temperature of lipids, Tm ~35°C, in vesicles prepared with a MFGM lipid extract. For temperature below Tm, atomic force microscopy revealed phase separation of lipids at 30 mN·m(-1) in Langmuir-Blodgett monolayers of the MFGM lipid extract. The high Tm lipids form liquid condensed (LC) domains that protrude by about 1.5 nm from the continuous liquid expanded (LE) phase. Cholesterol was added to the MFGM extract up to 30% of polar lipids (cholesterol/milk sphingomyelin (MSM) molar ratio of 50/50). Compression isotherms evidenced the condensing effect of the cholesterol onto the MFGM lipid monolayers. Topography of the monolayers showed a decrease in the area of the LC domains and in the height difference H between the LC domains and the continuous LE phase, as the cholesterol content increased in the MFGM lipid monolayers. These results were interpreted in terms of nucleation effects of cholesterol and decrease of the line tension between LC domains and LE phase in the MFGM lipid monolayers. This study revealed the major structural role of cholesterol in the MFGM that could be involved in biological functions of this interface (e.g. mechanisms of milk fat globule digestion).

The temperature-dependent physical state of polar lipids and their miscibility impact the topography and mechanical properties of bilayer models of the milk fat globule membrane

“…The characterization of high-Tm lipid rich domains below 35°C is also in agreement with previous studies performed with dried Langmuir-Blodgett monolayers [8,9]. By using the same MFGM lipid extract at 20°C, Murthy et al [8] showed the formation of liquid-condensed domains composed of high-Tm milk polar lipids protruding by about 1.5 nm from the liquid expanded phase composed by unsaturated phospholipids. By using milk phospholipids extracted from raw cream, other…”

“…The MFGM polar lipid extract used in this study was the same as in Murthy et al [8,23] Q water and adjusted to pH 6.7 using NaOH 5 M. PIPES 10 mM buffer with NaCl 50 mM and CaCl 2 2 mM (purity  99%; Sigma) was also prepared.…”

“…These polar lipids are characterized by a wide diversity of saturated and unsaturated acyl residues [7], with distinct melting temperatures (Tm) accordingly. High-Tm with a main phase transition at Tm = 36.4°C [8]. Using confocal laser scanning microscopy, lipid microdomains were observed in the external bilayer of the MFGM at the surface of native milk fat globules [9][10][11][12][13][14].…”

Differences in Interactions of Benzoic Acid and Benzoate with Interfaces

“…21,22,27,30–33 Although this system is powerful in providing the molecular detail, Langmuir monolayers of dipalmitoylphosphatidylcholine (DPPC) are frequently used models of eukaryotic cell membranes. Langmuir monolayers have been used previously for a variety of studies, detailing interactions of lipids with cholesterol, 34 phospholipids, fatty acids, 35 proteins, 36,37 and drugs. 38–41 By measuring the surface pressure of a phospholipid monolayer as a function of area per molecule, it is possible to compare a compound’s ability to interact with the phospholipid monolayer and determine if it is affecting the rigidity of the monolayer or the packing of the phospholipids.…”

Condensation of Model Lipid Films by Cholesterol: Specific Ion Effects

“…For instance, this technique has been used to bring new insight into the functional role of CHOL in milk polar lipids, used in the preparation of dairy emulsions, e.g., infant milk formulas. In particular, the role played by CHOL in the organization of biological membranes surrounding lipid droplets in milk, called the milk fat globule membrane (MFGM), has been recently investigated [30][31][32][33]. Accordingly, CHOL concentration facilitates the phase separation and reduces the mechanical resistance to rupture of coexisting phases in bilayer models of the MFGM.…”