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Colloids and Surfaces B: Biointerfaces 83 (2011) 29–41

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

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uman milk fat globules: Polar lipid composition and in situ structuralnvestigations revealing the heterogeneous distribution of proteins and theateral segregation of sphingomyelin in the biological membrane

hristelle Lopez ∗, Olivia MénardNRA, AGROCAMPUS OUEST, UMR 1253 Science et Technologie du Lait et de l’Oeuf, F-35000 Rennes, France

r t i c l e i n f o

rticle history:eceived 23 June 2010eceived in revised form 21 October 2010ccepted 21 October 2010vailable online 30 October 2010

eywords:ilk fat globule membrane

hospholipidphingomyelinytoplasmic crescent

a b s t r a c t

Although human milk fat globules (MFG) are of primary importance since they are the exclusive lipiddelivery carriers in the gastrointestinal tract of breast-fed infants, they remain the poorly understoodaspect of milk. The objectives of this study were to investigate these unique colloidal assemblies and theirinterfacial properties, i.e. composition and structure of their biological membrane. In mature breast milk,MFG have a mean diameter of 4–5 �m, a surface area of about 2 m2/g fat and an apparent zeta potential� = −6.7 ± 0.5 mV at 37 ◦C. Human MFG contain 3–4 mg polar lipids/g fat as quantified by HPLC/ELSD.The main polar lipids are sphingomyelin (SM; 36–45%, w/w), phosphatidylcholine (19–23%, w/w) andphosphatidylethanolamine (10–15%, w/w). In situ structural investigations of human MFG have beenperformed using light and confocal microscopy with adapted fluorescent probes, i.e. Nile Red, the extrinsicphospholipid Rh-DOPE, Fast Green and the lectin WGA-488. This study revealed a spatial heterogeneity

ipid domain in the human milk fat globule membrane (MFGM), with the lateral segregation of SM in liquid-orderedphase domains of various shapes and sizes surrounded by a liquid-disordered phase composed of theglycerophospholipids in which the proteins are dispersed. The glycocalyx formed by glycoproteins andcytoplasmic remnents have also been characterised around human MFG. A new model for the structureof the human MFGM is proposed and discussed. The unique composition and lateral organisation of thehuman MFGM components could be of metabolic significance and have health impact for the infants that

ed.

need to be further explor

. Introduction

Human milk is the natural and exclusive source of energynd biologically active molecules which are essential for optimalrowth and development of breast-fed infants in the initial phasesf their post-natal development and their performance as adults.lthough the chemical composition of human milk has been fullytudied and is thus quite well-known [1], information about thetructural organisation of its main components is scarce. Regard-ng more particularly the lipids, their organisation in the uniqueolloidal structures of milk fat globules (MFG) enveloped by a bio-ogical membrane remains the least understood aspect of milk. MFGre the lipid delivery carriers secreted by the mother for nutritional

urposes; they provide 40 up to 55% of total energy intake. More-ver, authors reported that breast MFG have a protective functionn the gastrointestinal tract of the newborn [2]. Hence, human MFGnd their surface properties need to be further investigated to bet-

∗ Corresponding author. Tel.: +33 2 23 48 56 17; fax: +33 2 23 48 53 50.E-mail address: Christelle.Lopez@rennes.inra.fr (C. Lopez).

927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2010.10.039

© 2010 Elsevier B.V. All rights reserved.

ter understand their functions in the digestive tract of infants andbe able to improve the preparation of infant formulas.

Human milk is an oil-in-water emulsion in which the organ-isation of lipids is complex and specific to this biological fluid.Human MFG are colloidal assemblies of about 4 �m diameter, thecore of which is mainly composed of triacylglycerols (98% of milklipids; [1]). The thin layer of membrane and membrane-associatedmaterial which surrounds the triacylglycerol-rich core of MFG com-monly is referred as the milk fat globule membrane (MFGM; [3]).Both the composition and the structure of the MFGM result fromthe mechanisms of secretion of MFG from the epithelial cells of themammary gland during the lactation period [3].

Human MFGM is mainly composed of proteins, glycoproteins,enzymes, polar lipids and cholesterol, which are typical con-stituents of cell membranes [3]. Recent publications reportedthe composition of human MFG proteins [4]. Among the human

MFGM proteins identified to date, the major proteins are xanthineoxidase, adipophilin, fatty acid binding protein and the heav-ily glycosylated proteins such as the mucins (mainly MUC-1 butalso MUC-4, MUC-15 . . .), lactadherin (PAS 6/7), CD36 (PAS 4),butyrophilin, some of them having multiple forms. The human

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FGM contains the same polar lipids as those present in theiological membranes of mammal species [5–8]. The glycerophos-holipids are phosphatidylcholine (PC), phosphatidylethanolaminePE), phosphatidylserine (PS), and phosphatidylinositol (PI). The

ain sphingolipid found in human milk is sphingomyelin (SM).lycosphingolipids which are quantitatively minor constituents of

he MFGM, comprise the cerebrosides (neutral glycosphingolipidsontaining uncharged sugars) and the gangliosides (acidic gly-osphingolipids containing sialic acid) [1].

The MFGM has gained a lot of attention recently, due to therowing interest in its nutritional, physiological and health prop-rties [9–11]. MFGM polar lipids have been reported to have variousenefits for human health due to their involvement in cell functionuch as growth and development, and transport systems [9,10].

hile MFGM proteins have a very low nutritional value, theylay important functions in defence mechanisms for the breast-ed infant and their bioactivity has been reviewed [10,11]. Authorsgree that the nutritional and health properties of the MFGM resultrom its chemical composition but they could also depend onhe lateral organisation of its constituents. However, few studiesnvestigated the organisation of the human MFGM in its nativenvironment, i.e. in situ in milk.

From a structural point of view, the organisation of the MFGMs a trilayer, a surface-active inner monolayer and an outerilayer membrane, is well-accepted [3]. Using electron microscopy,uthors observed that the first inner layer is an electron-denseaterial composed of proteins and polar lipids originating from

he endoplasmic reticulum which covers the surface of the lipidroplets within the cell [3]. The outer bilayer of the MFGM resultsrom the envelopment of lipid droplets in specialized regions of api-al plasma membrane of the mammary epithelial cells [3]. Authorslso observed using electron microscopy that the human MFGMas an externally disposed glycocalyx with numerous filamentseing oriented in the aqueous phase [12,13]. Using fluorescenceicroscopy, Evers et al. [14] recently showed heterogeneities in the

uman MFGM. From biochemical data and structural informationresent in the literature, authors proposed schematic represen-ations for the organisation of the human MFGM [4,8,14,15].owever, in all these speculative molecular architectures of theuman MFGM the lipid bilayer is presented as a neutral two-imensional solvent for membrane proteins, according to theinger and Nicholson fluid mosaic model proposed for biologicalembranes [16]. Up to date, authors did not consider the lateral

rganisation of polar lipids together with proteins to elucidate thetructure and function of the MFGM. On the basis of the recent char-cterisation of lipid domains in the bovine MFGM whatever the sizef MFG [17,18], the investigation of the lateral organisation of theuman MFGM appeared to be a stimulating research focus.

The objectives of this study were (i) to increase the knowl-dge about the ultrastructure and surface properties of humanFG, (ii) to determine the polar lipid composition of the MFGM

n mature breast milks and (iii) to investigate the lateral organisa-ion of polar lipids and proteins in the human MFGM surroundingat globules in their native environment, i.e. in situ in milk, usingonfocal microscopy and adapted fluorescent dyes. Such studiesbout breast MFG are of particular interest to improve the knowl-dge on the interfacial properties of natural biological assembliesompared to processed lipid droplets and for the development ofnnovative infant formulas.

. Materials and methods

.1. Human milk samples

Mature human milks have been collected from volunteerealthy women with 2–7.5 months post-partum, living in the West

s B: Biointerfaces 83 (2011) 29–41

part of France (French Britanny). Following their secretion from thelactating cells, expressed milks were removed from the mammarygland with a breast milk pump. The mature breast milks have beenstored at 4 ◦C and characterised within 24 h after their collection.

2.2. Human milk fat globule size measurements

The size distributions of human milk fat globules were measuredby laser light scattering using a Mastersizer 2000 (Malvern Instru-ments, Malvern, UK), equipped with a He/Ne laser (� = 633 nm)and an electroluminescent diode (� = 466 nm). The refractive indexof human milk fat was taken to be 1.460 at 466 nm and 1.458 at633 nm. The milks have been heated to 37 ± 1 ◦C before the exper-iments. About 0.2 mL of mature milk was diluted in 100 mL ofwater directly in the measurement cell of the apparatus in orderto reach 10% obscuration. The casein micelles were dissociated byadding 1 mL of 35 mM EDTA/NaOH, pH 7 buffer to the milks, inthe apparatus. The size distributions of human milk fat globuleswere characterised by the volume-weighted average diameter d43defined as

∑nid

4i/∑

nid3i, the volume-surface average diameter

d32 defined as∑

nid3i/∑

nid2i

where ni is the number of fat glob-ules of diameter di, and the modal diameter that corresponds tothe population of fat globules the most important in volume. Thespecific surface area was calculated by the software as S = 6·ϕ/d32,where ϕ is the volume fraction of milk fat. The experiments wereperformed in triplicate for each breast milk.

2.3. Apparent zeta potential measurements

Human milk fat globule electrophoretic mobility was mea-sured by electrophoretic light scattering using a Zetasizer 3000HS (Malvern Instruments) equipped with palladium electrodes andan avalanche photodiode detector. The apparent zeta-potential ofmilk fat globules was calculated as follows: the zeta-potential �of a particle is a value calculated from its electrophoretic mobil-ity �, according to Henry’s equation: � = (�·6��/ε)/f(a), where� and ε are the viscosity and dielectric constant of the solutionrespectively, at the temperature of the measurement. “1/” is theDebye length and “a” is the radius of the particle. The Smoluchowskiapproximation, assuming f(a) = 1.5, was used. The experimentshave been performed (i) at 20 ◦C, with � = 1.187 cp and ε = 79and (ii) at the physiological temperature 37 ◦C, with � = 0.77 cpand ε = 75. Samples were prepared by suspending 4–15 �L breastmilk in 10 mL of human milk ultrafiltrate in order to measurethe electrophoretic mobility of human milk fat globules in theirnative environment, devoid of proteins (caseins and whey pro-teins). � measurements have also been performed in a buffer(20 mM imidazole, 50 mM NaCl, 5 mM CaCl2, pH 7.0; � = 0.89 cp,ε = 79). All analyses were performed three times for each milksample.

2.4. Analysis of breast milk polar lipids

2.4.1. Chemicals, reagents and polar lipid standardsChloroform stabilized with ethanol (for analysis) and methanol

(HPLC grade) were purchased from Carlo Erba Reagents (Valde Reuil, France). Triethylamine (purity > 99%) and formic acid(purity > 98%) were purchased from Sigma–Aldrich (Saint QuentinFallavier, France). The polar lipid standards were supplied

phosphatidylethanolamine dipalmitoyl, N,N-dimethyl (C16:0);purity 99%), PI (l-� phosphatidylinositol ammonium salt from soy-bean; purity 98%), PS (1,2-diacyl-sn-glycero-3 phosphoserin; purity98%), PC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine; purity99%) and sphingomyelin (SM from bovine brain; purity 99%).

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.4.2. Extraction of total lipids and analysis of polar lipidsAn adapted protocol of the cold extraction procedure devel-

ped by Folch et al. [19] was used for the extraction of total lipidsrom the mature human milks, as detailed in Lopez et al. [20]. Totalipid extracts were stored at −20 ◦C until further analysis by higherformance liquid chromatography (HPLC).

The quantification of total polar lipids, e.g. glycerophospho-ipids and sphingomyelin, and the determination of the polar lipidlasses were performed using HPLC combined with an evapora-ive light scattering detector (ELSD). The chromatographic methodsed for the separation of the polar lipids was detailed in Lopezt al. [20]. The identification of the glycerophospholipids and thephingomyelin was carried out by a comparison with the retentionime of pure standards. To obtain a quantitative evaluation of thelycerophospholipids and sphingomyelin, five calibration curvesere determined from the area values obtained by injecting 10 �L

f chloroform:methanol (88:12, v/v) serially diluted solutions con-aining 0.25–2 �g of PE, 0.5–2.5 �g of PC, 0.25–2 �g of PS, 0.25–2 �gf PI and 0.5–2.5 �g of SM. Each solution was prepared and injectedn triplicate. The sum of glycerophospholipids (PE, PI, PS, PC) andphingomyelin concentration was regarded as total polar lipid con-entration in the human mature milks.

.5. Confocal laser scanning microscopy

The microstructural analysis of human milk fat globules waserformed as previously reported for bovine milk fat glob-les [17,18]. An inverted microscope NIKON Eclipse-TE2000-C1siNIKON, Champigny sur Marne, France) allowing confocal lasercanning microscopy (CLSM) and optical microscopy using differ-ntial interference contrast (DIC, also called Nomarski) was used.onfocal experiments were performed using an argon laser oper-ting at 488 nm excitation wavelength (emission was detectedetween 500 and 530 nm), an He-Ne laser operating at 543 nmavelength excitation (emission was detected between 565 and

15 nm) and a diode at 633 nm (detection with long pass > 650 nm).he observations were performed using a ×100 (NA 1.4) oil immer-ion objective.

The staining protocols of milk fat globules and of theFGM were developed in our laboratory and previously detailed

17,18]. Neutral lipids such as triacylglycerols were stainedith the lipid-soluble Nile Red fluorescent probe (5H-Benzo-phenoxazine-5-one, 9-diethylamino; Sigma–Aldrich, St Louis,SA), prepared with a concentration of 42 �g/mL. About 100 �Lf the Nile Red solution was added to 1 mL of breast milk.he fluorescent probe N-(Lissamine rhodamine B sulfonyl)ioleoylphosphatidylethanolamine (Rh-DOPE; Avanti polar lipids

nc, Birmingham, England) was used to label the phospholipids inhe human MFGM. About 20 �L of the commercial solution wasdded to 0.5 mL of breast milk. For the labelling of MFGM proteins,ast Green FCF (Sigma F7258, Sigma–Aldrich, St Louis, USA) wassed. Fast Green fluorescent probe was prepared by dissolving 1%f the powder in distilled water, and added to human milks at theoncentration of 10%. The lectin wheat germ agglutinin Alexa fluor88 (WGA; Invitrogen, Cergy Pontoise, France) was used to localisehe carbohydrate moities in the human MFGM. A 1 mg/mL solutionf WGA has been prepared by dissolving the powder in PBS buffer.2 M pH 7.4 (sigma P4417, Sigma–Aldrich, St Louis, USA). All theolutions containing the fluorescent probes were kept frozen untiltilisation. Low melting point agarose (Sigma, St Quentin Fallavier,rance) was prepared at 5 g/L and stored at 45 ◦C until utilisation.

After the step of labelling, the samples were kept at room tem-erature for at least 2 h before the microstructural analysis. Then,�L of the milk stained with the fluorescent probes was deposednto the glass, slowly mixed with 20 �L of the agarose and observedn the microscope. The microstructural analyses were performed at

s B: Biointerfaces 83 (2011) 29–41 31

room temperature (e.g. 19 ± 1 ◦C) and at the human physiologicaltemperature (e.g. 38 ± 1 ◦C), using a temperature-regulated stage(Linkam Scientific Instruments Ltd., Tadworth Surrey, England).

Differential interference contrast (DIC) microscopy was used tovisualize fat globules (localisation in milk, shape, size, presence ofcytoplasmic remnents). DIC images were sometimes superimposedto the emission fluorescent recorded in the CLSM images. The two-dimensional images had a resolution of 512 × 512 pixels and thepixel scale values were converted into micrometers using a scalingfactor. In the multiple labelled samples, different colours were usedto locate the fluorescent probes.

2.6. Statistical analysis

Analyses of variance (ANOVA) were performed using theGeneral Linear Model procedure of Statgraphics Plus version 5 (Sta-tistical Graphics Corp., Englewood Cliffs, NJ). Differences betweenthe treatment means were compared at the 5% level of significanceusing Fisher’s least significance difference (LSD) test.

3. Results and discussion

3.1. Human milk fat globules: size distribution and apparent zetapotential

As observed using optical and fluorescence microscopy, lipidsare present in human milk in the form of spherical dropletswith polydisperse sizes (Fig. 1). These droplets correspond tothe supramolecular assemblies called the human milk fat glob-ules (human MFG) which are secreted by the epithelial cellsof the mother to vehicle the triacylglycerols and other bioac-tive molecules (fat-soluble vitamins, phospholipids, sphingolipids,cholesterol, MFGM proteins) in the gastrointestinal tract of thenewborn. Triacylglycerols, that represent about 98% of human milklipids, have been labelled using Nile Red as hydrophobic lipid sol-uble fluorescent probe (Fig. 1B). The overlay of the images takenby light microscopy with DIC (Fig. 1A) and by confocal microscopyusing Nile Red (Fig. 1B) showed that the triacylglycerols are exclu-sively located in the core of human MFG (Fig. 1C). Human MFGare enveloped by a biological membrane, the MFGM, the structureof which has been investigated using CLSM and adapted fluores-cent probes as reported above. The diffusion of Nile Red fromthe aqueous phase of milk to the triacylglycerol-rich core of MFGshowed that the MFGM is permeable to some molecules and thatexchanges are possible between the interior and the exterior ofhuman MFG.

Laser light scattering measurements showed that maturehuman MFG have a bimodal size distribution ranging from about0.35 to 13 �m (Fig. 1). The size parameters calculated from thehuman MFG size distributions of the mature milks expressed byfive different mothers are reported in Table 1 and compared tothe data reported in the literature. Whatever the stage of lacta-tion (from 2 to 7.5 months post-partum), human milk lipids weredispersed in MFG with a diameter of about 4.5–5 �m (Table 1). Thesurface area of human MFG suspended in the surrounding aqueousphase was about 2 m2/g of fat, as calculated from the MFG size dis-tributions (Table 1). These results are in accordance with previousresults reported in the literature [15,21].

The apparent zeta potential � of human MFG as determined intheir native environment was � = −6.7 ± 0.5 mV at 37 ◦C (physio-logical temperature) and � = −7.6 ± 0.6 mV at 20 ◦C (Table 1). These

results are in agreement with the � values previously determinedat room temperature in a buffer [15]. The negative charge of humanMFG is carried out by some of the polar lipids and proteins located inthe MFGM, in interaction with the minerals present in the aqueousphase surrounding fat globules. The absolute value of �-potential

32 C. Lopez, O. Ménard / Colloids and Surfaces B: Biointerfaces 83 (2011) 29–41

Fig. 1. Microstructure of mature human milk. Size distribution of human milk fat globules determined using laser light scattering and observed using (A) light microscopywith differential interference contrast (DIC; also called Nomarski) and (B) confocal laser scanning microscopy with the labelling of triacylglycerols performed with Nile Redfluorescent dye. (C) Overlay of DIC and confocal images showing the localisation of triacylglycerols in the core of human milk fat globules.

Table 1Structural parameters and apparent zeta potential of human milk fat globules (mean ± standard deviation; n = 4–8 independent milks per mother for the experimental data).

Human milks

Experimental data (2–7.5 months post-partum) Literature

Mother #A Mother #B Mother #C Mother #D Mother #E [21] [15]

Size parametersMode (�m) 5.1 ± 0.3 5.0 ± 0.1 4.5 ± 0.2 4.9 ± 0.1 4.2 ± 0.2 ND NDd32 (�m) 3.2 ± 0.2 3.5 ± 0.1 3.1 ± 0.2 3.4 ± 0.1 3.1 ± 0.1 4.0 ± 0.3 3.5 ± 0.1d43 (�m) 5.1 ± 0.4 5.2 ± 0.1 4.6 ± 0.2 5.2 ± 0.1 4.3 ± 0.1 5.2 ± 0.4 4.4 ± 0.2Surface (m2/g fat) 2.0 ± 0.1 1.9 ± 0.1 2.1 ± 0.1 1.9 ± 0.1 2.1 ± 0.1 1.4 ± 0.1 1.9 ± 0.1

Apparent zeta potential (mV)at 20 ◦C in HM-UF ND −7.6 ± 0.5 −7.6 ± 0.7 ND −7.6 ± 0.6 ND NDat 37 ◦C in HM-UF ND −6.6 ± 0.5 −6.7 ± 0.5 ND −6.7 ± 0.6 ND NDat 20 ◦C in buffer ND −6.3 ± 0.3 −6.2 ± 0.2 ND −6.4 ± 0.1 ND −7.8 ± 0.1at 37 ◦C in buffer ND −5.2 ± 0.5 −5.3 ± 0.2 ND −5.3 ± 0.3 ND ND

Abbreviations: d43 = nid4i/nid

3i; d32 = nid

3i/nid2

iwhere ni is the number of fat globules of diameter di . Surface = 6·ϕ/d32, where ϕ is the volume fraction of milk fat;

HM-UF = human milk ultrafiltrate (aqueous phase of human milk, without proteins and lipids); ND = non determined.

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etermined for human MFG is lower than the �-potential valuesetermined for cow and buffalo MFG [17,18,22].

.2. Phospholipid and sphingomyelin composition of the humanFGM

The human milk polar lipid composition has been investigatedn mature milks expressed by three different mothers (2–5 monthsost-partum; exclusive breastfeeding). Fig. 2 shows representativeigh-performance liquid chromatograms of polar lipids obtainedy elution of total lipid extracts from the mature human milks.he well-separated HPLC peaks correspond to the five major polaripid classes found in human milk: PE, PI, PS, PC and SM (Fig. 2).hese polar lipids are mainly located in the MFGM surrounding fatlobules. As shown in Fig. 2, PE, PI and PS eluted each as a singlePLC peak whereas PC eluted as 4 HPLC peaks that may corre-

pond to PC species with different fatty acid compositions and SM

luted as 5 HPLC peaks that may differ both from their sphingoidase and from their fatty acid composition (Fig. 2). The relative

ntensity of the HPLC peaks recorded for PC and SM changed asfunction of the mother who expressed the mature milk (Fig. 2).sing the same HPLC/ELSD method, our group previously reported

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ig. 2. Normal-phase liquid chromatography (LC) evaporative light-scatteringetector (ELSD) chromatograms of the total lipid fraction obtained from matureuman milks expressed by 3 mothers, as indicated in the figure. Each chromatogram

s representative of the chromatograms obtained for the mature milks from the sameother (two to three independent milks collected per mother, two extractions of

at, three injections). The polar lipids from the human milk lipid globule membranere phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserinePS), phosphatidylcholine (PC) and sphingomyelin (SM).

able 2olar lipid composition of human milks (mean ± standard deviation; n = 3).

Polar lipids

Experimental data

Mother #A Mother #B Moth

Polar lipids (%)PE 15.1 ± 1.0 10.1 ± 1.6 14.3PI 9.5 ± 1.1 11.7 ± 0.5 9.2PS 15.6 ± 0.1 12.3 ± 0.8 18.4PC 23.5 ± 0.5 20.4 ± 2.1 19.3SM 36.4 ± 1.9 45.5 ± 2.6 38.8

Polar lipids (PL; mg per g of fat)PE 0.61 ± 0.05 0.31 ± 0.06 0.51PI 0.39 ± 0.04 0.36 ± 0.02 0.33PS 0.64 ± 0.03 0.37 ± 0.02 0.65PC 0.96 ± 0.07 0.63 ± 0.10 0.69SM 1.49 ± 0.11 1.38 ± 0.04 1.34Choline-containing PL 2.44 ± 0.17 2.01 ± 0.12 2.04Total PL 4.08 ± 0.21 3.05 ± 0.20 3.52

L: polar lipids, PE: phosphatidylethanolamine, PI: phosphatidylinositol, PS: phosphatidya PS + PI; Choline-containing polar lipids: sum of PC and SM; ND: Non determined.

s B: Biointerfaces 83 (2011) 29–41 33

4 HPLC peaks for PC and 3 HPLC peaks for SM from buffalo andbovine milks [18,20,22], the relative proportion of PC species beingaffected by the diet [20]. The HPLC signature is different for humanSM compared to bovine SM and buffalo SM, revealing differencesin the molecular species (sphingoid base and fatty acids). The HPLCprotocols used by Hundrieser and Clark [7] and van Beusekomet al. [23] did not allow the separation of several peaks for PCand SM, nor the experiments which have been performed usingthin layer chromatography [6,8]. Further experiments are requiredto identify the PC and SM molecular species present in maturehuman milks.

The total amount and relative proportion of each species of polarlipids are reported in Table 2. The mature human milks containedabout 3–4 mg polar lipids per g of fat (0.3–0.4%, w/w; Table 2). Thisis in agreement with previous data reported in the literature sincethe range is 0.3–1% (w/w) of mature milk lipids [1]. Bitman et al.[6] reported that polar lipids correspond to 0.4–0.5% (w/w) of totallipids in human milk during most of lactation. There is relatively fewdata on human milk polar lipid composition and the discrepanciesfound in the literature may result from differences in the size ofMFG and to the analytical methods used.

Among the different polar lipid species, SM was the main polarlipid of mature human milks (Table 2). This is in agreement withexisting literature on class distribution of human milk polar lipids[5,6,8], although authors reported that PC is the main polar lipid inhuman milk [7]. By the addition of PC and SM, we calculated thatabout 1.6–2.5 mg choline-containing polar lipids per g of fat werepresent in the mature human milks (Table 2). The high amounts ofcholine-containing polar lipids found in the human MFGM are ofparticular importance for the infants since choline is required forrapid organ growth and membrane biosynthesis in neonates. Usingthe same HPLC/ELSD method, we found more SM in the maturehuman milks compared to bovine and buffalo milks [18,20,22].This suggests that the relative proportion of each class of polarlipids may be adapted to the needs of the newborns from speciesto species.

3.3. Human milk fat globules: in situ structural investigations

3.3.1. Lateral segregation of polar lipids in the human MFGM:

presence of liquid-ordered phase domains rich in sphingomyelin

The lateral organisation of polar lipids (e.g. SM, PE, PC, PI, PS)in the biological membrane surrounding human MFG was investi-gated in situ in mature breast milks using CLSM and an extrinsicphospholipid fluorescently head-labelled with rhodamine (Rh-

Literature

er #C [5] [6] [7] [8]

± 2.2 27.7 19.3 23.8 ± 3.3 21.3 ± 4.7± 0.6 5.4 6.1 5.3 ± 3.0± 0.7 9.3 8.8 3.7 ± 1.5 16.4 ± 3.9a

± 2.4 24.9 28.4 33.2 ± 5.5 19.0 ± 2.2± 4.5 32.4 37.5 29.0 ± 6.4 43.3 ± 2.6

± 0.16 ND ND ND ND± 0.07 ND ND ND ND± 0.11 ND ND ND ND± 0.20 ND ND ND ND± 0.12 ND ND ND ND± 0.31 ND ND ND ND± 0.63 5.5 4–5 ND ND

lserine, PC: phosphatidylcholine, SM: sphingomyelin.

34 C. Lopez, O. Ménard / Colloids and Surfaces B: Biointerfaces 83 (2011) 29–41

F mbranfl o the lo milk fi egend

DfipguwadtsRwaMotidaidttnnflM

ig. 3. CLSM images showing the phospholipids in the human milk fat globule meuorescent domains (black colour) observed around milk fat globules correspond tf fat globules contains triacylglycerols. (C) Three-dimensional characterisation ofndicated in the figure. (For interpretation of the references to colour in this figure l

OPE). Fig. 3 shows the emission fluorescence of Rh-DOPE in theorm of red rings at the periphery of human MFG characterisedn their equatorial section which correspond to the labelling ofolar lipids in the MFGM. The fluorescent probe Rh-DOPE inte-rated the human MFGM, as previously reported for bovine MFGMsing the same methodology [17,18]. The interior of human MFG,hich is mainly composed of triacylglycerols as shown in Fig. 1,

ppears as non-fluorescent black areas in the CLSM images. Three-imensional observations of human MFG have been performed byhe recording of thin optical sections at different z-depths of milkamples (Fig. 3C). CLSM experiments revealed heterogeneities inh-DOPE emission fluorescence in the human MFGM. Domainsithout emission fluorescence of Rh-DOPE, corresponding to the

bsence of the probe and appearing as black areas in the humanFGM, were dispersed in the surrounding emission fluorescence

f Rh-DOPE (Fig. 3). These domains have been characterised in theop and bottom parts of human MFG and in their equatorial plane asndicated by arrows in Fig. 3B. Fig. 4 shows that the non-fluorescentomains located in the MFGM surrounding human MFG are char-cterised by various sizes and complex patterns with circular orrregular shapes. In Fig. 4B, the non-fluorescent areas are indepen-ent domains with a circular shape and a diameter from about 0.3o 1.3 �m. In Fig. 4A and C, some non-fluorescent domains seem

o be connected to each other by narrow non-fluorescent chan-els as pointed out by small arrows. In Fig. 4D and E, elongatedon-fluorescent domains, more or less connected to form someower-like shapes are observed in the outer bilayer of the humanFGM.

e thanks to their labelling with Rh-DOPE fluorescent probe (red colour). The non-ateral segregation of sphingomyelin in liquid-ordered phase domains. The interiorat globules performed by the recording of optical sections at different z-depths, as, the reader is referred to the web version of the article.)

These non-fluorescent domains which have been revealed usingRh-DOPE as MFGM probe did not correspond to the location ofMFGM proteins as discussed above. These non-fluorescent domainswere interpreted as preferential packing of sphingolipids (mainlySM) leading to their lateral separation from the glycerophospho-lipids (PC, PE, PI, PS) in the plane of the human MFGM, as previouslyreported for bovine MFGM [17,18]. This lateral heterogeneity in theorganisation of polar lipids in the human MFGM is generated on thebasis of differential lipid–lipid interactions and results from the dif-ferent structural properties and fatty acid compositions betweensphingolipids and glycerophospholipids. In mature human milks,Bitman et al. [6] reported that SM is rich in saturated long-chainfatty acids (16:0, 18:0, 20:0, 22:0, 23:0, 24:0) characterised by highmelting temperatures whereas the glycerophospholipids PC, PE, PSand PI are rich in 18:0 and in the kinked unsaturated fatty acylchains 18:1 c9 and 18:2 c9c12 (n-6) that have low melting temper-atures and are responsible for the fluidity of the biomembrane. As aconsequence of its sphingoid base and saturated fatty acid composi-tion, SM has specific physical properties which support a structuralrole for SM in maintaining MFGM bilayer stability and rigidity [6].As reported in the literature, sphingolipids can segregate togetherwith cholesterol in the plane of biological membranes to form lipiddomains in the liquid ordered (Lo) phase, the most actively studied

of which being the lipid rafts [24]. The surrounding glycerophos-pholipids (PC, PE, PI, PS) are in the fluid liquid disordered (Ld)phase [24]. As all the biological membranes, human MFGM containscholesterol (10–20 mg/dl; [1]) that can tightly pack with SM to formdomains in the Lo phase. The size of these Lo phase domains rich

C. Lopez, O. Ménard / Colloids and Surfaces B: Biointerfaces 83 (2011) 29–41 35

Fig. 4. Microstructural analysis of the human milk fat globule membrane (MFGM) performed in situ in mature breast milks using CLSM and the extrinsic head-labelledphospholipid Rh-DOPE. (A) CLSM image showing a human milk fat globule in the equatorial plane with its triacylglycerol (TAG) core and the surrounding MFGM withnon-fluorescent domains as indicated by arrows. (A–E) CLSM images showing the lateral segregation of sphingomyelin (SM) and cholesterol in liquid-ordered phase domainsof various sizes and shapes (non-fluorescent domains of black colour) dispersed in the human MFGM. These SM-rich domains were observed thanks to the preferentialp rophod terprew

idTtdRdoShgnMrlt

h(vpwlbMb

artitioning of Rh-DOPE fluorescent probe in the liquid disordered matrix of glyceomains are indicated by small arrows. Scale bars are indicated in the figure. (For ineb version of the article.)

n SM and cholesterol formed in the human MFGM allowed theirirect observation at a microscopic scale using CLSM (Figs. 3 and 4).hese Lo phase domains have been revealed by the selective par-itioning of the fluorescent probe Rh-DOPE for the fluid Ld phaseue to its unsaturated N-acyl chains (two 18:1 c9 chains in DOPE).h-DOPE did not integrate the tightly and densely packed Lo phaseomains enriched in SM and cholesterol, leading to the observationf non-fluorescent domains in the CLSM images (Figs. 3 and 4). SinceM corresponds to about 35–45% (w/w) of polar lipids in matureuman milks (Table 2), it is quantitatively possible that its segre-ation in Lo phases together with cholesterol corresponds to theon-fluorescent domains characterised in the outer leaflet of theFGM surrounding human MFG (Figs. 3 and 4). Moreover, authors

eported that SM, PC and the glycolipids are located in the outerayer of the MFGM while PE, PS and PI are mainly concentrated onhe inner surface of the MFGM [25].

Heterogeneities in the lateral organisation of polar lipidsave been revealed in the MFGM surrounding each human MFGFigs. 3 and 4). Moreover, differences have been observed betweenarious MFG from the same mature breast milk (Figs. 3 and 4), asreviously reported [14]. The morphology of the SM-rich domains,

hich was revealed by the partitioning of Rh-DOPE delimiting Lo

ipid phase boundaries (Figs. 3 and 4), is the result of a balanceetween line tension and dipole–dipole repulsion. In the bovineFGM, authors characterised Lo phase domains as dynamic assem-

lies with a circular shape corresponding to fluid-like domains

spholipids (emission fluorescence; red colour). The channels connecting SM-richtation of the references to colour in this figure legend, the reader is referred to the

[17,18]. The differences in the morphology of the SM-rich Lo

phase domains observed between bovine and human MFGM couldbe interpreted as differences in SM composition (sphingoid baseand/or fatty acid), SM concentration, cholesterol concentration andSM/cholesterol ratio between bovine and mature breast milk.

The thermotropic behaviour of MFGM lipid domains was stud-ied. Similar Lo phase domains were characterised in human MFGMat room temperature and at the physiological temperature, e.g.36–38 ◦C, showing that the lateral segregation of SM could persistat the temperature of milk digestion in the gastrointestinal tract ofthe newborns.

Although the coexistence of sphingolipids and glycerophos-pholipids in the MFGM is well-accepted from a chemical pointof view, several authors proposed their homogeneous distribu-tion in the outer bilayer of the MFGM [4,8,10,14,15]. Such arandom organisation of polar lipids in the MFGM correspondsto the Singer–Nicholson fluid mosaic model that proposes thatthe lipid bilayer of cell biological membranes functions as a neu-tral two-dimensional solvent with lipids in the liquid disorderedstate having little influence on membrane protein function [16].According to our results on the human MFGM (this paper) and pre-

vious work on the bovine MFGM [17,18], the presence of Lo phasedomains rich in SM and cholesterol dispersed in the fluid Ld matrixcomposed of the glycerophospholipids support the concept of thenon-random lateral organisation of polar lipids in the MFGM ofvarious mammal species.

3 urfaces B: Biointerfaces 83 (2011) 29–41

qcahcrtltctcldworgi(

tmFhemflbolilctlJo

3h

gpatptispposRrh

ilClpsh

Fig. 5. CLSM images of human milk fat globules showing the heterogeneous distri-bution of proteins, glycoproteins and polar lipids in the milk fat globule membrane(MFGM). (A and B) Heterogeneous distribution of the MFGM proteins characterisedin the equatorial plane of fat globules; proteins are pointed by arrows, triacylglyc-erols (TAG) are located in the core of fat globules. (C and D) Fluorescent labelling ofproteins and polar lipids in the MFGM, (C) proteins are labelled with fast green, (D)co-localisation of polar lipids (red colour) and proteins (blue colour) in the MFGMshowing the heterogeneous distribution of proteins (pointed by arrows) and thelateral segregation of sphingomyelin in liquid-ordered phase domains of variousshapes (non-fluorescent domains; black colour). (E and F) Fluorescent labelling ofglycoproteins and proteins in the MFGM surrounding a human milk fat globule, gly-coproteins are labelled with WGA-488 (E) and total proteins are labelled with Fast

6 C. Lopez, O. Ménard / Colloids and S

The SM-rich Lo phase domains revealed around MFG raise theuestion of the presence of lipid rafts in the human MFGM. Theoncept of “raft” refers to a particular kind of domains. A currentlyccepted definition of rafts is: small (10–20 nm), heterogeneous,ighly dynamic, sterol- and sphingolipid-enriched domains thatompartmentalise cellular processes [26]. Simons and Ikonen [27]eported that in polarised epithelial cells, lipid rafts accumulate inhe apical plasma membrane. Since MFG are secreted by the epithe-ial cells of the human mammary gland after being enveloped byhe apical plasma membrane [3], the lipid domains rich in SM andholesterol characterised in the human MFGM could be assimilatedo lipid rafts. However, (i) the size of the SM-rich Lo phase domainsharacterised around human MFG in situ in mature breast milks wasarger than the definition proposed by Pike [26], and (ii) the lateraliffusion of the SM-rich domains in the plane of the human MFGMas not observed, in contrast to Lopez et al. [17]. From the lipid rafts

f the apical plasma membrane to the SM-rich Lo phase domainsevealed in the human MFGM, structural and compositional reor-anisations may have occurred (i) during the envelopment of MFGn the apical plasma membrane leading to their secretion, and/orii) post-secretion.

From a methodological point of view, the use of CLSM to inves-igate the organisation of the human MFGM in situ in breast

ilks appeared to be highly relevant. Using DiIC18(3)-DS andM4-64 as lipophilic probes, Evers et al. [14] previously revealedeterogeneous fluorescence in the human MFGM suggesting het-rogeneities in the structure and composition. Although theseolecules are not phospholipids, Evers et al. [14] argued that these

uorescent probes were located in the outer layer of the MFGMilayer of phospholipids. As special care is needed for the selectionf fluorescent lipid probes, we decided since 2007 to use the head-abelled extrinsic phospholipid Rh-DOPE as a probe that resemblests natural counterpart, i.e. milk phospholipids, to investigate theateral organisation of polar lipids in the MFGM [17,18] and inomplex dairy products [28]. This methodological choice permittedo reveal the lateral segregation of SM from the glycerophospho-ipids (PC, PE, PI, PS) in the plane of the MGM. The group of Primenez-Flores (CalPoly, USA) recently confirmed the pertinencef our strategy by investigating the bovine MFGM [29].

.3.2. Simultaneous localisation of polar lipids and proteins in theuman MFGM

The organisation of proteins in the human MFGM was investi-ated in situ in mature breast milks using Fast Green as fluorescentrobe. Fig. 5A and B shows that some proteins are distributed inthin layer surrounding MFG while other proteins exhibit a large

hickness in the equatorial section of MFG and are distributed asatches (pointed by arrows in Fig. 5). Regarding the top and/or bot-om view of human MFG, proteins are heterogeneously distributedn the MFGM (proteins are pointed by arrows in Fig. 5C and D) andome domains of the MFGM are not fluorescently labelled by therotein probe (Fig. 5C), nor by the simultaneous labelling of theroteins and polar lipids (Fig. 5D). According to the size and shapef these non-fluorescent domains, we interpreted that they corre-pond to the SM-rich Lo phase domains previously described usingh-DOPE as fluorescent probe (Fig. 4). Hence, CLSM experimentsevealed that the SM-rich Lo phase domains characterised in theuman MFGM did not contain proteins.

Since glycosylated molecules are important regarding their rolen the protection of breast-fed infants [2,13], we investigated theocalisation of glycoproteins and glycolipids in the human MFGM.

LSM experiments were performed using the fluorescently labelled

ectin wheat germ agglutinin (WGA-488) which is a sugar-bindingrotein. Using the dual labelling Fast green and WGA-488, wehowed that glycoproteins are located at the external periphery ofuman MFG and that they only correspond to a part of total MFGM

green (F); TAG are located in the core of fat globules. Scale bars are indicated in thefigure. (For interpretation of the references to colour in this figure legend, the readeris referred to the web version of the article.)

proteins (Fig. 5E and F). For the simultaneous labelling of the gly-cosylated molecules and polar lipids located in the human MFGM,we used WGA-488 and Rh-DOPE probes as previously developedfor the characterisation of the bovine MFGM [17,18]. Fig. 6 showsthe co-localisation of glycosylated molecules and polar lipids in thehuman MFGM surrounding MFG. Fig. 6A and B shows the top viewof human MFG with the heterogeneous distribution of the glyco-sylated molecules and the polar lipids laterally segregated to formthe SM-rich domains. Fig. 6C shows a human MFG in the equa-torial section with its triacylglycerol-rich core surrounded by theMFGM containing the phospholipids and patches of glycosylated

molecules protruding in the aqueous phase of milk. The three-dimensional characterisation of human MFG was performed byrecording images at different z-depths from their top to their equa-torial section (Fig. 6D), as previously reported [17]. The emission

C. Lopez, O. Ménard / Colloids and Surfaces B: Biointerfaces 83 (2011) 29–41 37

Fig. 6. CLSM images showing the heterogeneous distribution of glycosylated molecules (yellow/green colour) and phospholipids (red colour) in the human milk fat globulemembrane (MFGM). Glycosylated molecules were stained using the lectin WGA-488 as fluorescent probe while phospholipids were stained using the head-labelled extrinsicphospholipid Rh-DOPE. (A and B) Top views of milk fat globules showing the glycosylated molecules and the liquid ordered domains that correspond to the lateral segregationo colouo at difft eader

floicpomet(spbtsma

mWdtalCiDL

f sphingomyelin (SM) from the other polar lipids (non-fluorescent domains, blackf glycoproteins in the MFGM. (D) Thin sections of milk fat globules characterisedhe figure. (For interpretation of the references to colour in this figure legend, the r

uorescence of WGA-488 was recorded until 2 �m over the surfacef some MFG, showing that some glycoproteins highly protruden the aqueous phase surrounding human MFG to form the glyco-alyx (Fig. 6C and D). This is in agreement with previous studieserformed using electron microscopy, that reported the presencef extended filamentous structures mainly composed of the higholecular weight glycoproteins such as the mucin MUC-1 which

xtend from the surface of human MFG [2,12,13]. Since WGA selec-ively binds to N-acetylglucosamine and N-acetylneuraminic acidsialic acid) residues and according to the carbohydrate compo-ition of the human MFGM glycoproteins, we deduced that therobe selectively bound at least to the mucins (MUC-1), lactadherin,utyrophilin, and CD36 which are the main glycoproteins charac-erised in the human MFGM [4]. Glycosphingolipids that containialic acid (N-acetylneuraminic acid) as part of their carbohydrateoiety, which are called gangliosides, are also labelled with WGA

s previously reported in the literature [14,17].These CLSM experiments performed in situ in mature breast

ilks revealed that the human MFGM contains (i) manyGA-receptors corresponding mainly to glycoproteins, that are

istributed heterogeneously on the surface of MFG and protrude inhe aqueous phase surrounding MFG to form the glycocalyx (Fig. 6Cnd D), (ii) SM-rich Lo phase domains that do not contain glycosy-

ated molecules which appear as non-fluorescent domains in theLSM images (Fig. 6A and D), and (iii) glycoproteins and/or glycol-

pids located at the boundary of the SM-rich domains (Fig. 6A and). The lack of proteins and glycosylated molecules in the SM-rich

o phase domains is in agreement with previous studies investi-

r). (C) Milk fat globule in the equatorial plane showing phospholipids and patcheserent z-depths from the surface to the equatorial plane. Scale bars are indicated inis referred to the web version of the article.)

gating the structure of the MFGM in situ in milk using fluorescencetechniques [14,17,18]. We can emit the hypothesis that the SM-rich Lo phase domains could contribute to the lateral segregationof proteins in the MFGM.

3.3.3. Cytoplasmic remnents connected to human milk fatglobules

The microstructural analysis of mature breast milks performedusing light microscopy with differential interference contrast (DIC)revealed the presence of biological entities attached to some MFG(Fig. 7A, D and G). These MFG were characterised further usingconfocal microscopy with various fluorescent probes (Fig. 7). Thestaining of breast milks using Nile Red as hydrophobic lipid fluores-cent probe revealed the labelling of the triacylglycerol core of MFG(Fig. 7B) and the non-labelling of the attached entity as showed bythe overlay of DIC and CLSM images (Fig. 7C). Hence, we concludedthat the entities were attached to MFG and that they did not cor-respond to partial coalescence of MFG nor to non-spherical MFG.CLSM experiments performed using Rh-DOPE as fluorescent probeshowed emission fluorescence in the MFGM surrounding MFG andin the entities (Fig. 7E and F), suggesting that the entities wereenveloped by phospholipids and/or that they were rich in phos-pholipids (Fig. 7D–I). On the basis of our CLSM experiments and

data reported in the literature, we interpreted these biological enti-ties as being cytoplasmic remnents from lactating cells connectedto some human MFG. Indeed, authors reported that in some occa-sion the mechanisms involved in the closure of the apical plasmamembrane behind the projecting lipid droplet lead to their secre-

38 C. Lopez, O. Ménard / Colloids and Surfaces B: Biointerfaces 83 (2011) 29–41

Fig. 7. Human milk fat globules bearing cytoplasmic remnents (CR) characterised both by light and by confocal microscopy with adapted fluorescent probes. (A, D and G)Images of light microscopy with differential interference contrast (DIC, also called Nomarski) showing CR connected to milk fat globules. (B, E and H) Confocal laser scanningm ydropa holipa

t[wt

stccaauntMMmMpcnwdm

icrographs showing the emission fluorescence of (B) Nile Red used to stain the hnd (E and H) Rh-DOPE, the head-labelled fluorescent probe used to stain the phosprrows. The scale bars are indicated in the figure.

ion with a piece of cytoplasm remaining attached to MFG in milk3,30]. Authors reported that cytoplasmic remnents can be filledith intracellular membranous matter [14], which could explain

heir labelling by Rh-DOPE (Fig. 7D–I).Cytoplasmic remnents were observed around MFG of various

izes, as shown in Fig. 7. Their volume varied from one human MFGo another (Fig. 7). As shown in Fig. 7, the cytoplasmic materialan exceed the human MFG volume. The morphology of adheringytoplasmic remnents looked like a crescent around fat globules,s previously reported [30]. These structural observations are ingreement with the experiments performed by Evers et al. [14]sing DiIC18-DS as fluorescent probe to label the cytoplasmic rem-ents. Even if cytoplasmic remnents were characterised in each ofhe mature breast milks used in this study, the great majority of

FG did not contain cytoplasmic remnents (Fig. 1). Main humanFG are enveloped compactly by the MFGM, as a result of theechanisms of their secretion (Fig. 3). Regarding the amount ofFG with cytoplasmic remnents, authors reported that their pro-

ortion varies between and within species [30]. Human milk can

ontain until about 7–30% of fat globules with cytoplasmic rem-ents, while cow milk contains the least amount of fat globulesith crescents (1%) [30]. This could explain the reason why weid not observe fat globules with cytoplasmic remnents in cowilk [17,18].

hobic lipids such as triacylglycerols (TAG) located in the core of milk fat globulesids. (C, F and I) Overlay of the light and fluorescence images. The CR are pointed by

3.4. Human MFG are unique lipid delivery carriers covered by abiomembrane with a specific composition and structure: newinsights

3.4.1. Proposed model for the structure of the human MFGMFrom our structural investigations which have been performed

using CLSM in situ in mature breast milks, we propose a new modelfor the structure of the human MFGM (Fig. 8). The human MFGMhas a complex architecture in which the polar lipids are the back-bone. Polar lipids are organised in the MFGM as a trilayer withthe polar head groups exposed to an aqueous environment (cyto-plasm, aqueous phase of milk) and the hydrocarbon tails forminga hydrophobic area in the centre of the bilayer or in contact withthe triacylglycerol core. Moreover, and for the first time, the modeldescribes the coexistence of at least two lipid phases in the outerbilayer of the human MFGM: (i) the Ld phase composed of the glyc-erophospholipids (PE, PC, PI, PS) and (ii) the lateral segregation ofSM in Lo phase domains (Fig. 8). The kinked structure of the unsat-urated fatty acyl chains of glycerophospholipids results in a shorter

molecular length compared to the straight SM molecules that havelong-chain saturated fatty acids and a sphingoid base. Hence, thereis probably an interdigitation among the tails of the SM moleculesin the MFGM bilayer and cholesterol can fill the voids between theheads of SM. The presence of SM-rich Lo phase domains across the

C. Lopez, O. Ménard / Colloids and Surfaces B: Biointerfaces 83 (2011) 29–41 39

Fig. 8. Structure of the human milk fat globule membrane (MFGM). The trilayer of polar lipids is the backbone of the MFGM, with a lateral organisation in the plane of thebilayer corresponding to the phase separation of sphingomyelin (SM) and cholesterol in liquid-ordered (Lo) phase domains. The glycerophospholipids (phosphatidylcholine,PC; phosphatidylethanolamine, PE; phosphatidylserine, PS; phosphatidylinositol, PI) are organised in a liquid-disordered (Ld) phase surrounding the SM-rich Lo phasedomains. The lipid bilayer in SM-rich L phase domains can be symmetric (SM in the two leaflets of the bilayer) or asymmetric (SM in the outer layer of the MFGM) and theseL e spaca ed aloo e, in th

tte[tdp5dps

3ur

la.cMtetgpMaie

o

o phase domains can induce local higher thickness of the MFGM. Cholesterol fills thpposing layer. The MFGM transmembrane and peripheral bound proteins are locatf glycoproteins and glycolipids are distributed over the external membrane surfac

wo leaflets of the MFGM bilayer (symmetric bilayer) or only onhe outer leaflet of the MFGM (asymmetric bilayer) remains to belucidated, as already discussed for bovine MFGM in Lopez et al.18]. Local differences in the thickness of the MFGM can exist dueo the presence of SM that associate laterally to form rigid Lo phaseomains. Indeed, authors reported that Lo phase domains rich in SMrotrude from the background of glycerophospholipids by about–9 A [31]. CLSM experiments revealed that the SM-rich Lo phaseomains are devoid of proteins and glycosylated molecules (glyco-roteins, glycolipids), that remain in the Ld phase of the MFGM, aschematically presented Fig. 8.

.4.2. Human milk fat globules: colloidal assemblies with anique structure and composition providing specific functionaloles in the infant digestive tract

Milk fat globules provide a convenient system for deliveringarge amounts of energy and essential compounds (essential n-6nd n-3 fatty acids, cholesterol, polar lipids, fat-soluble vitamins. .) in the gastrointestinal tract of the suckling neonate. Hence, weould consider that the composition but also the structure of humanFG have been optimised through the selective pressure to assure

he protection, nutrition and optimal growth of newborns. How-ver, all the properties of MFG have not yet been revealed, mainlyhe non-nutritive ones. The challenge is to understand the potentialastrointestinal functions that the size of MFG and their interfacial

roperties, both the composition and the lateral organisation of theFGM components, could provide to the infant. On the basis of the

nalytical results and in situ structural investigations performedn this study, several main points regarding human MFG appearssential to be highlighted and are discussed above.

e under the head groups of SM or extends the interdigitating fatty acyl chain in theng the membrane in the Ld phase matrix of phospholipids. The glycosylated moities

e glycocalyx. The drawing is highly schematic and the sizes are not proportional.

3.4.2.1. Size of human milk fat globules and surface area covered bythe MFGM. The hydrophobic nature of triacylglycerols, which arethe second largest component of human milk (3–5 g per 100 mL),prevents their solubility in the aqueous phase of milk [2]. Hence,the purpose of the natural compartmentalisation of milk lipids inthe form of fat globules with a mean diameter around 4–5 �m and alarge surface area covered by the MFGM (about 2 m2/g of fat, Table 1and Fig. 1) is:

(i) to permit the solubilisation of triacylglycerols in milk, to facili-tate their hydrolysis by the digestive lipases (lingual and gastriclipases, pancreatic lipase and the bile salt-stimulated lipasepresent in breast milk), the efficient absorption of fatty acidsand monoacylglycerols and their metabolisation by breast-fedinfants [2].

(ii) to increase the bioavailability of nutrients required for infantgrowth and development. For example, the bioavailability offat-soluble vitamins (A, D, E, K) and sterols (10–20 mg/dl inhuman milk; [1]), particularly the cholesterol that is located inthe outer bilayer of the MFGM.

(iii) to favour the interaction between MFGM components andpathogenic microorganisms in the gastrointestinal tract for theprotection of the infants toward infections [2].

3.4.2.2. Unique composition and structure of the human MFGM: high

amount of SM leading to the formation of Lo phase domains. The nutri-tional and health significance of the high SM content in human milk,e.g. 35–45% of polar lipids, should be further explored because ofthe bioactive properties of dietary sphingolipids and their hydrol-ysis products (sphingosines and ceramides) [9,10]. Particularly, SM

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0 C. Lopez, O. Ménard / Colloids and S

as been reported to be involved in neonatal gut maturation duringhe suckling period [32] and myelination of the developing centralervous system [9,10].

The high amount of SM also plays a structural role in the MFGM.or the first time to the author’s knowledge, the phase separation ofolar lipids in the human MFGM leading to the lateral segregationf SM into Lo phase domains that do not contain proteins nor glyco-ylated components was revealed (Figs. 5 and 6). These SM-rich Lo

hase lipid domains could have unique biophysical properties, dif-erent from those of the fluid Ld matrix of the MFGM, which coulde involved in biological processes occurring in the gastrointestinalract of the infant. Regarding fat digestion process, the size of MFGnd the specific structural organisation of the MFGM may play aey role in the mechanisms of milk lipid hydrolysis and absorption33]. Since the lipid–water interface is essential for the adsorb-ion of lipases and their activity, we emit the hypothesis that theresence of SM-rich Lo phase domains acting as rigid platforms athe surface of the human MFGM (Figs. 3–6) may be of functionalignificance in the mechanisms of MFG digestion. This need to beurther explored, as already discussed for bovine MFGM [17,18].he preferential localisation of the digestive lipases in the humanFGM (i) on the rigid SM-rich Lo phase domains, (ii) on the fluid

d matrix of glycerophospholipids or (iii) at the junction betweenhe Lo and the Ld phase domains needs to be elucidated in situ in

ilk. These questions are of the utmost importance since authorsuggested that the type of polar lipids present at the surface ofipid droplets may govern lipase activity in the digestive tract andhowed that SM decrease the level of gastric lipolysis comparedo the glycerophospholipids [33]. Furthermore, the changes in the

FGM surface topography during the hydrolysis of triacylglycerolore by the digestive lipases should be investigated in situ in milksing microscopy techniques. Particularly, the effect of the gastric

ipase should be explored since it is essential for the subsequentydrolysis of breast MFG by pancreatic and human milk bile salt-timulated lipases [34].

.4.2.3. Heterogeneous distribution of oligosaccharide-containingolecules at the surface of human MFG: a physical barrier to protect

he newborn against the microorganisms. CLSM experiments per-ormed in this study using the lectin WGA as a fluorescent probeevealed, in situ in breast milk, that human MFG are coated withlycosylated molecules (i.e. glycosylated proteins and glycosylatedipids) (Fig. 6). The glycoproteins were heterogeneously distributedt the surface of the human MFGM and protruded in the aqueoushase surrounding fat globules. The important role played by non-

mmunological components such as oligosaccharide-containingolecules (mucins, lactadherin, gangliosides, cerebrosides) located

t the surface of the human MFGM has been well documented. Theyave been reported to provide biological, physiological and protec-ive functions in the gastrointestinal tract of the breast-fed infantsrom enteric and other pathogens [10,35]. Oligosaccharides derivedrom the mammalian epithelial cells of the mother such as somelycoproteins (mucins, lactadherin) and glycolipids (gangliosides,articularly GM1) found in the human MFGM provide a defensivetrategy acting as decoys to prevent adhesion of enteric pathogensnd viruses to the epithelial cells of the infant’s gut, thereby protect-ng beast-fed infants against infections [13]. Butyrophilin, which islso a major glycoprotein of the human MFGM has no known pro-ective function in breast milk [13]. Moreover, Buchheim et al. [12]rgued that the high molecular weight glycoproteins forming thelycocalyx around human MFG may enhance triacylglycerol diges-

ion efficiency and release of free fatty acids by binding the digestiveipases.

.4.2.4. Presence of cytoplasmic remnents. Biological entities cor-esponding to cytoplasmic remnents have been observed around

s B: Biointerfaces 83 (2011) 29–41

human MFG (Fig. 7). Cytoplasmic remnents contain nearly allintracellular membranes and organelles of the milk-secreting cell,except nuclei [3]. Even if cytoplasmic crescents have no nutritionalvalue, they represent an important route of cellular substancesinto milk, such as mRNAs, enzymes, micronutrients, hormones andgrowth factors. They can also be viewed as a source of foreign anti-gens from the mother that may have some conditioning effect onthe developing immune system of the newborn [3]. The apparentevolutionary persistence of cytoplasmic remnents on human MFGsuggests that they may have beneficial effects in the young [3].

3.4.2.5. Natural colloidal assemblies of fat globules in breast milkvs. processed lipid droplets in infant formulas. Human MFG arebiophysical systems designed by the mammary gland to delivernutrients as well as both nutritive and non-nutritive messagesto the neonate during suckling. The size of human MFG and theunique characteristics of the MFGM, both its composition and itsstructure, provide specific properties to breast milk as compared tothe processed lipid droplets of infant formulas. The design of infantformulas aims at mimicking human milk from a compositionalpoint of view to provide the required nutrients to the infants.However, the structure of the components, particularly the lipids,is not yet considered by the industrials. Using high-pressurehomogenisation, lipids (mainly of vegetable origin) are emulsifiedin tiny droplets, e.g. 0.3–0.5 �m mean diameter (surface area∼30 m2/g fat), covered by milk proteins (caseins, whey proteinsor hydrolysed proteins) and other surface-active molecules suchas phospholipids (i.e. soya lecithin) [36]. Such drastic differencesin the suprastructure and interfacial properties of processed lipiddroplets vs. native breast MFG raise questions about the conse-quences on (i) the lost of the specific protective functions of theMFGM, (ii) the mechanisms of lipid digestion and (iii) the nutrition,development, growth and health of infants. New strategies shouldbe developed by tailoring lipid droplets that resemble breast milkfat globules, in order to improve infant formulas. For example,some MFGM-based ingredient concentrated from by-productssuch as bovine buttermilks could be added to infant formulas as afunctional ingredient in order to benefit the specific composition,structure and functions of the MFGM [10].

4. Conclusions

This study provides new information about human MFG. Thein situ structural investigations of mature breast milks performedusing CLSM and pertinent fluorescent probes allowed (i) the local-isation of triacylglycerols in the MFG core and (ii) the localisationof polar lipids, proteins and carbohydrate moities of glycoproteinsand glycolipids in the surrounding MFGM. Moreover, for the firsttime, this study reveals the lateral segregation of sphingomyelinin Lo phase domains which are surrounded by a Ld phase matrixof glycerophospholipids in which the proteins and glycosylatedmolecules are heterogeneously dispersed. Sphingomyelin, whichaccounts for 35–45% of mature breast milk polar lipids, providesspecific properties to the MFGM that need to be further investi-gated. The composition and structure of the human MFGM shouldbe characterised during early lactation, in the colostrum secretedat 1–5 days and transitional milk until about 15 days post-partum.Increasing the knowledge about the structure and composition ofthe unique delivery systems that are the human milk fat globulesis of utmost interest regarding the potential applications for theimprovement of formulas providing health benefits for the infants.

Acknowledgements

The authors thank the volunteer mothers for donations of milksamples. Halima Sahraoui, Agathe Bouchoux, Floriane Mercier and

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olan Pradeau are greatly acknowledged for their active partici-ation in this scientific research project. F. Rousseau (INRA UMRTLO, Rennes, France) is acknowledged for help in the apparent zetaotential measurements. The authors thank Viviane Picard (INRAMR STLO, Rennes, France) for help in bibliographic research.

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