The miscibility of milk sphingomyelin and cholesterol is affected by temperature and surface pressure in mixed Langmuir monolayers

Highlights

• The interactions between milk sphingomyelin and cholesterol are involved in the formation of membrane domains.

• The melting temperature of milk-SM is Tm = 34 °C.

• Below Tm, cholesterol has a weak condensing effect on milk-SM.

• Above Tm, cholesterol and milk-SM are miscible with strongest area condensation for 30 mol% chol.

• The physical state of milk-sphingomyelin controls its assembly with cholesterol.

Abstract

The miscibility of milk sphingomyelin (milk-SM) and cholesterol was investigated in this study. The effect of different physical states of milk-SM on its interactions with cholesterol was determined by the recording of isotherms of compression of Langmuir films for temperatures above and below the gel to Lα phase transition of milk-SM (T_m ∼ 34 °C). For T = 15 °C < T_m, the liquid expanded (LE) to liquid condensed (LC) phase transition of milk-SM monolayers was observed at surface pressures of 10–15 mN/m. For T = 43 °C > T_m, the milk-SM molecules were in a LE phase regardless of the surface pressure applied. A phase diagram pressure – milk-SM/cholesterol composition was established. This study demonstrated that both temperature and surface pressure affected the miscibility between the milk-SM and cholesterol. The strongest attractive forces (i.e. condensing effect) were identified for 30 mol% cholesterol when the milk-SM was in the LE phase state.

Introduction

The biological membrane surrounding milk fat globules is currently poorly understood despite its importance in the functional properties of milk lipids, in the mechanisms of milk lipid digestion and in nutritional and health benefits. Increasing the knowledge surrounding the packing and miscibility of lipid components (i.e. polar lipids, cholesterol) is of primary importance in order to better understand e.g. the functions of the milk fat globule membrane (MFGM) and the properties of emulsions containing lipid droplets coated with milk polar lipids.

The composition and architecture of the MFGM result from the mechanisms of fat globule secretion from the mammary epithelial cells (Keenan & Mather, 2006). The MFGM contains membrane-specific proteins and a highly complex assortment of polar lipids: sphingolipids (mainly sphingomyelin, milk-SM: 20–45% of polar lipids depending on mammal species), phosphatidylcholine (PC: 19–23.7%), phosphatidylethanolamine (19.3–32.7%), phosphatidylserine (1.9–19.7%) and phosphatidylinositol (6.1–13.6%) (Lopez, 2011, Rombaut et al., 2007). Apart from these lipids, the MFGM also contains about 30 wt% of cholesterol in the membrane lipid fraction (Mesilati-Stahy & Argov-Argaman, 2014 – i.e. about 45 mol%). The MFGM is structured as a trilayer of polar lipids and proteins (Keenan and Mather, 2006, Lopez, 2011). The inner monolayer is in contact with the triacylglycerol core of the fat globules and originates from the endoplasmic reticulum of the epithelial cells. The outer bilayer envelops the fat globules during their secretion through the apical plasma membrane of the mammary cells. Previous authors have reported an asymmetry in the localization of polar lipids with milk-SM, PC and cholesterol being preferably located in the outer bilayer of the MFGM (Deeth, 1997). Studies performed in situ in milk by confocal microscopy have revealed a phase separation of polar lipids in the outer bilayer of the MFGM with the formation of lipid domains (Gallier et al., 2010, Lopez et al., 2010, Lopez and Ménard, 2011, Nguyen et al., 2016, Zou et al., 2015). These lipid domains were assumed to be formed by the lateral segregation of saturated polar lipids with a high phase transition temperature, mainly milk-SM, and could also contain cholesterol (Gallier et al., 2010, Lopez, 2011, Lopez et al., 2010, Nguyen et al., 2016). The role played by cholesterol in the formation and biophysical properties of milk-SM rich domains in the outer bilayer of the MFGM is still poorly understood (Murthy et al., 2016a, Murthy et al., 2015). Recent studies performed by atomic force microscopy have shown that cholesterol affects the morphology of milk-SM domains (Guyomarc'h et al., 2014, Murthy et al., 2015, Murthy et al., 2016a), and reduces the resistance to perforation of the membrane bilayers when studied as models of the MFGM (Guyomarc'h et al., 2014, Murthy et al., 2016a). Also, compression isotherms of the MFGM polar lipid monolayers revealed the condensing effect of the cholesterol, with concomitant changes in the topography of the Langmuir-Blodgett monolayers (Murthy et al., 2015). The structural and functional roles of cholesterol in the MFGM remain poorly understood (Guyomarc'h et al., 2014, Murthy et al., 2015, Murthy et al., 2016a), despite cholesterol being known to play a fundamental role in the organization of cell membranes. Cholesterol has been reported to be involved in several mechanisms due to its close packing association with saturated lipids, inducing phase separation (McMullen et al., 2004, Quinn and Wolf, 2009, Wolf et al., 2001) and forming tightly packed microdomains in membranes called “rafts”, specifically with sphingolipids (Ramstedt and Slotte, 1999, Simons and Ikonen, 1997). The role played by the milk-SM in the activity of the gastric lipase at the surface of lipid droplets has been demonstrated (Favé et al., 2007) and the role of cholesterol and physical state of SM in the hydrolysis rate of SM by sphingomyelinase has been reported (Contreras et al., 2004, Jungner et al., 1997, Ruiz-Argüello et al., 2002). The biophysical properties of the lipid domains present in the MFGM could therefore modulate the activity of lipolytic enzymes during milk fat globule digestion. Authors have recently shown that milk-SM improves lipid metabolism in high fat diet-fed mice (Norris, Jiang, Ryan, Porter, & Blesso, 2016) and that the milk-SM is involved in the reduction of cholesterol absorption in the intestine (Eckhardt et al., 2002, Noh and Koo, 2004, Nyberg et al., 2000). Further studies are necessary to better understand the interactions between the milk-SM and cholesterol that could be involved in several functions, particularly in the gastrointestinal tract. These studies need to be performed under experimental conditions adapted to the biological conditions. Most of the biophysical experiments reported in the scientific literature were performed at room temperature (i.e. 20 °C). However, temperature governs the physical state of the milk-SM (temperature of phase transition T_m ∼ 34 °C; Murthy et al., 2015) and MFGM polar lipids (Murthy, Guyomarc’h, & Lopez, 2016b) and milk lipids are digested at 37 °C in the gastro-intestinal tract, i.e. at a temperature above the T_m of milk-SM.

The objective of this work was to determine whether the milk-SM and cholesterol were miscible and how temperature influenced their miscibility. The original approach taken in this study was to investigate the effect of the phase state of milk-SM molecules, through changes in temperature (i.e. T < T_m or T > T_m of milk-SM), on the interactions between milk-SM and cholesterol. The thermotropic phase behaviour of the milk-SM was determined by differential scanning calorimetry and X-ray diffraction. The miscibility of cholesterol and the milk-SM was investigated by the isotherms of compression of Langmuir monolayers of several milk-SM/cholesterol mixtures. For the first time, this work demonstrated the condensing effect of cholesterol on milk-SM monolayers with a maximum area of condensation recorded for 30 mol% cholesterol when the milk-SM was in the LE phase state.