Phospholipids A K H MacGibbon, Fonterra Research Centre, Palmerston North, New Zealand M W Taylor, Massey University, Palmerston North, New Zealand ª 2011 Elsevier Ltd. All rights reserved.
Introduction
Structure
Although polar lipids are only a very small proportion of the lipids in milk, they play an important role due to their mixed hydrophilic and hydrophobic nature. Their dual hydrophilic and hydrophobic structure contributes largely to stabilization of the suspension of milk fat in the aqueous environment of liquid milk, thus allowing the relatively high concentrations of milk fat and protein to coexist in the same solution. This is accomplished by the integral part that polar lipids play in the milk fat globule membrane (MFGM) and, to a lesser extent, the general emulsification properties in the aqueous solution. The major structural features involved are the large nonpolar (hydrophobic) fatty acid chains and the polar (hydrophilic) charged head group residues of the phospholipids.
The structures of polar lipids found in milk are shown in Figures 1 and 2. Phosphatidic acid has fatty acids at positions sn-1 and sn-2 and a phosphate group at the sn3 position. The glycerol phospholipids (e.g., phosphatidylcholine and phosphatidylethanolamine) are based on phosphatidic acid with a moiety attached to the phosphate at the sn-3 position. Plasmalogens (not shown in the figures) have a similar structure to phosphatidylcholine and phosphatidylethanolamine but with an ether linkage rather than an ester linkage at the sn-1 position. Lysophospholipids have only one fatty acid in the glycerophospholipid. These are the products of phospholipases, enzymes that can degrade phospholipids. The sphingophospholipid, sphingomyelin, consists of a ceramide (a fatty acid linked to a sphingosyl base through an amide linkage) and a phosphorylcholine group. Sphingomyelin is usually included as a phospholipid. The ionic properties of phospholipids are important in their function as emulsifiers. Table 2 shows the typical ionization constants of glycerophospholipids, illustrating the charge state of the various phospholipids. At the normal pH of milk (pH 6.7), the properties can range from a zwitterion, with no net charge (phosphatidylcholine, phosphatidylethanolamine), to an acid with a net negative charge (phosphatidylserine, phosphatidylinositol). Glycoceramides (glycosphingolipids) have one or more hexose sugar units attached at the 1-position of the ceramide (Figure 2). Gangliosides are complex ceramide polyhexosides, which contain one or more sialic acid groups (N-acetylneuraminic acid (NANA)). The specific names of the gangliosides identify their structure (the letter G followed by M, D, T, or Q designates the mono-, di-, tri-, or quatrasialic acid group and the number indicates the carbohydrate sequence that is attached to the ceramide (5 n, where n is the number of neutral sugar residues)). For example, GM3 is a ganglioside with one NANA unit and two neutral sugar residues (Figure 2). A number of gangliosides have been isolated from bovine milk (GM3, GM2, GM1, GD3, GD2, GD1). The major gangliosides are GD3 (50%) and GM3 (20%) (Figure 2).
General Features Lipids are usually classified on the basis of polarity as neutral lipids or polar lipids. However, an alternative classification often quoted is ‘simple lipids’ and ‘complex lipids’ (simple lipids yield only two types of primary products on hydrolysis, while complex lipids yield three or more primary products). Under this second definition, polar lipids are complex lipids while neutral lipids are simple lipids. Polar lipids can contain a variety of polar groups that contribute to the charged nature of the molecule. Phospholipids contain a charged phosphate group (Figure 1), whereas glycolipids contain polar carbohydrate groups, which increase the solubility in water (Figure 2). While phospholipids are the major polar lipid components of milk, glycolipids also need to be considered as they have important biological functions, especially in nerve and brain function. In humans, they are involved in a number of disease states such as Tay–Sachs disease, which is characterized by an enzyme deficiency causing the accumulation of a specific ganglioside. Common abbreviations for polar lipids are given in Table 1.
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Figure 1 Phospholipid structures. R9 and R0 are fatty acids at sn-1 and sn-2 positions, respectively.
Source The MFGM that surrounds the milk fat droplets is derived from the apical plasma membrane of secretory cells in the lactating mammary gland. The MFGM is composed of phospholipids and glycolipids as well as protein, glycoprotein, enzymes, triacylglycerols, and minor components. Estimates of the proportion of phospholipids in the MFGM vary from 15 to 30%. About 60–65% of the phospholipids in milk are associated with the MFGM. The remaining 35–40% are found in the aqueous phase associated with protein/membrane fragment material in solution rather than still attached to the MFGM. Polar lipids constitute about 0.5–1% of total milk lipids. This percentage of phospholipids does vary a little with the stage of lactation. Phospholipid levels in milk tend to decline during lactation, although they can increase near the end. While there is a small change in the percentage of
phospholipids, the ratio of major phospholipids remains relatively constant, suggesting a constant ratio in the MFGM. Table 3 shows the percentage of polar lipids in bovine milk. Phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin are the major components and they contribute equally to the total polar lipid content. Phosphatidylserine and phosphatidylinositol are present at lower levels and there are only trace (but important) amounts of ceramides and gangliosides. As the milk is processed, the phospholipids are partitioned differently from the neutral lipids (Table 4). When the whole milk is separated, the phospholipids tightly associated with the MFGM go into the cream with the neutral lipids, while those associated with the protein/membrane fragments in the aqueous phase are retained in the skim milk. Hence, the phospholipids-to-total fat ratio is greater in whole milk than in cream. Furthermore, during buttermaking, a greater proportion of the phospholipids than the
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neutral lipids from the cream are retained in the buttermilk, leading to a high ratio of phospholipids to total fat.
Chemical Properties
Figure 2 Structure of gangliosides (GM3 and GD3).
Table 1 Common abbreviations for polar lipids Abbreviation
Full name
PC PE PS PI PA Sph (or SM) CMH CDH NANA MFGM
Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Sphingomyelin Ceramide monohexoside Ceramide dihexoside N-acetylneuraminic acid (sialic acid) Milk fat globule membrane
The positional distribution of fatty acids in the major phospholipids of bovine milk (phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) is shown in Table 5. Unlike triacylglycerols, phospholipids do not have short-chain fatty acids (C14:0 being the shortest chain fatty acid that is significant). In glycerophospholipids, the unsaturated fatty acids, especially the polyunsaturated fatty acids, tend to be preferentially esterified at the sn-2 position, while the saturated fatty acids are at the sn-1 position. There is a range of degree of unsaturation among the specific phospholipids. Phosphatidylethanolamine has a high content of unsaturated fatty acids, especially linoleic acid and even linolenic acid, far higher than that found in the milk fat triacylglycerols. The unsaturated fatty acids are found predominantly at the sn-2 position, while the C18:1 is fairly evenly distributed and the C18:0 and C16:0 are at the sn-1 position. On the other hand, phosphatidylcholine is more saturated than phosphatidylethanolamine, and the distribution of saturated and unsaturated fatty acids is less distinct between the sn-1 and sn-2 positions, although the C18:0 and the polyunsaturated fatty acids still show the preference described above. Sphingomyelin has a different structure (Figure 1). The major fatty acid moiety is either C16:0 or a longerchain fatty acid (C22:0 to C24:0), producing an almost completely saturated fatty acid composition. However, the major sphingoid base is sphingosine (C18:1), which introduces unsaturation into the molecule.
Analysis Milk phospholipids can be extracted with slightly polar solvents such as methanol, ethanol, or chloroform.
Table 2 Typical ionization constants (pKa) of the polar groups of phospholipids, ionic strength 0.1 NaCl
Ionizable group Phosphate Phosphate (2) Carboxylic Ammonium pH range of neutral charge Type Values determined in 0.1 mol l
Phosphatidyl Phosphatidic choline Phosphatidylethanolamine Phosphatidylserine Phosphatidylinositol acid 1.0
1.7
2.6
3–12
11.2 4–9
5.5 11.5 4
Zwitterion
Zwitterion
Weakly acidic
1
2.5
3.0 8.0
Acidic
Acidic
NaCl. Values will be altered for higher ionic strength or in emulsions and membranes.
Milk Lipids | Phospholipids Table 3 Polar lipid composition of bovine milk a
Polar lipid
Percentage of total polar lipids
Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Phosphatidylinositol Sphingomyelin Plasmalogens Ceramides Gangliosides
34.5 31.8 3.1 4.7 25.2 3.0 Trace Trace
a Polar lipids account for 0.5–1% of total lipids in milk. From Patten and Jensen (1976).
composition of glycerophospholipids can be determined by fatty acid methyl ester (FAME) analysis, as for triacylglycerols. The fatty acids are hydrolyzed from glycerol, esterified, and then analyzed by gas chromatography. However, the fatty acid of the sphingophospholipid sphingomyelin is more difficult to release due to the attachment by an amide bond and requires extensive acid hydrolysis prior to methylation of the released fatty acid.
Features
Acetone is usually a poor solvent for phospholipids and can be used for precipitation and concentration. Thin-layer chromatography is a simple and useful technique for the qualitative determination of phospholipid groups. With the use of appropriate stains and standards, the position of most of the specific phospholipids can be determined. More quantitative analysis can be carried out by high-performance liquid chromatography (HPLC) and mass spectroscopy. The fatty acid
Dairy phospholipids are of major importance because they are able to stabilize emulsions and foams, and contribute to the formation of micelles and membranes. Phospholipids also have the potential to be prooxidants, because they contain mono- and polyunsaturated fatty acids and have the ability to attract metal ions. Mono- and polyunsaturated fatty acids and metal ions are known to accelerate lipid oxidation, especially when heat is applied; hence, the phospholipids can be lost
Table 4 Approximate lipid content of different milk products
Product
Total fat wt%
Phospholipids wt%
Phospholipids/total fat %
Whole milk Skim milk Cream 40% Buttermilk
4 0.06 40 0.6
0.035 0.015 0.21 0.13
0.9 25 0.5 22
Adapted from Mulder H and Walstra P (1974) The Milk Fat Globule. Farnham Royal, UK: Commonwealth Agriculture Bureaux.
Table 5 Fatty acid composition of the major phospholipids of bovine milk Phosphatidylcholine
Phosphatidylethanolamine
Fatty acid
sn-1
sn-2
sn-1
sn-2
Sphingomyelina,b
14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4 22:0 23:0 23:1 24:0 25:0
5.6 41.9 0.6 17.5 20.3 2.7 0.8
10.8 30.6 1.2 2.4 27.8 9.2 1.8 1.6 1.2
1.9 19.7 1.2 19.0 45.8 2.9 1.1 0.2 0.2
1.3 4.7 2.2 1.3 47.8 21.4 4.5 2.2 3.0
2.6 22.9 0.8 4.7 5.2 0.9
0.2
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15.2 28.0 1.0 15.4 0.6
a From Morrison WE, Jack EL, and Smith LM (1965) Fatty acids of bovine milk glycolipids and phospholipids and their specific distribution in the diacylglycerophospholipids. Journal of the American Oil Chemical Society 42: 1142–1147. b Fatty acid linked to amide nitrogen of the base.
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during processing. However, in dairy products, the situation is complex and it appears that the phospholipids act as both prooxidants and antioxidants, depending on pH and the water content and phospholipid species. In addition to their importance in cell membranes and cell signaling, specific polar lipids are reputed to have a number of positive health effects related to immune system, heart health, brain health, and cancer. These are related to either the polar lipids themselves or their breakdown products. Sphingomyelin, gangliosides, phospholipids, and plasmalogen are implicated in different aspects of prevention of cancer. Further research will elucidate whether dairy polar lipids have unique properties related to their fatty acid composition or are simply another source of generic polar lipids. See also: Milk Lipids: General Characteristics; Milk Fat Globule Membrane; Nutritional Significance.
Further Reading Bitman J and Wood DL (1990) Changes in milk fat phospholipids during lactation. Journal of Dairy Science 73: 1208–1216. Christie WW (2003) Lipids Analysis : Isolation, Separation, Identification and Structural Analysis of Lipids, 3rd edn. Bridgwater, England: Oily Press. Christie WW, Noble RC, and Davies G (1987) Phospholipids in milk and dairy products. Journal of the Society of Dairy Technology 40: 10–12.
Dewettinck K, Rombaut R, Thienpont N, Trung Le T, Messens K, and Van Camp J (2008) Review –Nutritional and technological aspects of milk fat globule membrane material. International Dairy Journal 18: 436–457. Fong BY, Norris CS, and MacGibbon AKH (2007) Protein and lipid composition of bovine milk-fat-globule membrane. International Dairy Journal 17: 275–288. Gunstone FD (2008) Phospholipid Technology and Applications. Bridgwater, England: The Oily Press. Gunstone FD, Harwood JL, and Dijkstra AJ (2007) The Lipid Handbook. London: Chapman & Hall. Karlsson AA, Arnoldsson KC, Westerdahl G, and Odham G (1997) Common molecular species of glucosyl ceramides, lactosyl ceramides and sphingomyelins in bovine milk determined by highperformance liquid chromatography–mass spectrometry. Milchwissenschaft 52: 554–559. MacGibbon AKH and Taylor MW (2006) Composition and structure of bovine milk lipids. In: Fox PF and McSweeney PLH (eds.) Advanced Dairy Chemistry, Vol. 2: Lipids, 3rd edn., pp. 1–42. New York: Springer. Marsh D (1990) CRC Handbook of Lipid Bilayers. Boca Raton, FL: CRC Press. Morrison WE, Jack EL, and Smith LM (1965) Fatty acids of bovine milk glycolipids and phospholipids and their specific distribution in the diacylglycerophospholipids. Journal of the American Oil Chemical Society 42: 1142–1147. Mulder H and Walstra P (1974) The Milk Fat Globule. Farnham Royal, UK: Commonwealth Agriculture Bureaux. Olsson NU and Salem N (1997) Review: Molecular species analysis of phospholipids. Journal of Chromatography B 692: 245–256. Parodi PW (1997) Cows’ milk fat components as potential anticarcinogenic agents. Journal of Nutrition 127: 1055–1060. Patton S and Jensen RG (1976) Biomedical Aspects of Lactation. New York: Pergamon. Ward RE, German JB, and Corredig M (2006) Composition, applications, fractionation, technology and nutritional significance of milk fat globule membrane material. In: Fox PF and McSweeney PLH (eds.) Advanced Dairy Chemistry, Vol. 2: Lipids, 3rd edn., pp. 213–244. New York: Springer.