DAIRY FOODS
Lipids of Bovine and Human Milks: A Comparison 1 ROBERT G. JENSEN, ANN M. FERRIS, CAROL J. LAMMI-KEEFE, and ROBIN A. HENDERSON Department of Nutritional Sciences University of Connecticut Storrs 06269-4017 ABSTRACT
ing. In our first review on human milk lipids, we observed that sampling had been poorly done and that modern methods of analysis had not always been used (41). Fortunately, this has changed and now there are reliable data on the c~mposition of human milk. In this paper, we wIll compare data on the lipids in bovine and human milks. Blanc (3) and Renner (58) have presented reviews on the comparison. Other reviews that we have used are by Christie (12, 13) and Jensen and Clark (36) on bovine milk lipids and by Jensen (38) on human milk lipids. A short version of this review has been presented (38). This paper is not intended to be comprehensive.
Human and bovine milks contain about 3 to 5% total lipid, existing as emulsified globules 2 to 4 Jlm in diameter and coated with a membrane derived from the secreting cell. About 98% or more of the lipid is triacylglycerol, which is found in the globule. Phospholipids are about .5 to 1% of total lipids and sterols are .2 to .5%; these are mostly located in the globule membrane. Cholesterol is the major sterol. The major differences are in fatty acid composition, triacylglycerol structure, and the response of fatty acids in human milk to changes in diet. Bovine milk contains substantial quantities of 4:0 to 10:0, about 2% 18:2, and almost no other long-chain polyunsaturated fatty acids. The fatty acid composition is not altered by ordinary changes in diet. Human milk contains very little 4:0 to 10:0, 10 to 14% 18:2, and small quantities of other polyunsaturates. The triacylglycerol structure differs, with much of the sn-2 position occupied by 16:0 in human milk and 4:0 to 10:0 at sn-3 in bovine milk. The effects of milk cholesterol and fatty acids on human blood cholesterol levels are discussed. (Key words: lipids in milk, cholesterol)
THE NATURE OF MILKS
INTRODUCTION
Lipids of human milk have not been investigated as extensively as those of bovine milk. However, this is changing, because research on bovine milk lipids is almost at a standstill, but investigations of human milk lipids are inereas-
Received June 22. 1989. Accepted August 18. 1989. lScienti~C Contri~ution Nwnber 1295. StOrTS Agricultural Ellperunent Stauon, The University of Connecticut. StOrTS 06269-40 17. 1990 ] Dairy Sci 73:223-240
We have listed in Table 1, (3, 32, 46) the general composition of human and bovine milks. The data in Table 1 represent an oversimplification of the complexity of milk, but note that human milk contains less protein and ~ore lactose than bovine milk. More profound dIfferences include the higher nonprotein nitrogen content of human milk. The figures are averages for human milk, which change as lactation progresses. Bovine milk is consistently uniform because the milk pooled for processing is usually homogenized and colostrum is excluded. With human milk, each sample from an individual at a particular time can differ in content and composition from previous samples or from those of other women otherwise matched for time, age, diet, etc. (32, 36, 41). Almost always the infant receives an individual feeding of unprocessed human milk, which is unique in itself. Compared with bovine milk, volumes of human milk are small, up to 100 mllfeeding with an average of about 750 mIld for mature milk (34). Many factors can affect the volume and composition of human milk, the most influential being time postpartum (stage of lactation) and diet. These influences are essentially elimi-
223
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JENSEN ET AL.
TABLE 1. General composition (%) of human and bovine milks. t Casein % of
Milk
Protein
protein
Fat
Lactose
TOlal solids
Ash
Kilocalories
12.1
.2
12.7
.7
72 58 75
(%)
Human Mature. 36 d Colostrum, 3 d Bovine
1.0 2.3 3.4
40 82
3.9 3.0 3.7
6.8 5.5 4.8
1Adapled from (3. 32. 46).
nated in bovine milk by pooling. In Table I, the protein content of human colostrum is higher than in mature milk, 2.3 vs. 1.0%, and the fat and lactose lower, 3.0 vs. 3.9% and 5.5 vs. 6.8%. The volume of colostrum is also lower. The biosynthetic pathways for bovine and human milk lipids are essentially identical. The major exceptions are: 1) bovine rumen activity, which provides short-chain fany acids, 4:0 to 10:0, biohydrogenates dietary polyunsaturated fatty acids (PUFA), decreasing the amounts of these in milk and producing trans and positional isomers of unsaturates, and limiting the effects of dietary changes on the composition of bovine milk fatty acids; and 2) differences in acylglycerol transferases, which result in species specific patterns of triacylglycerol (TG) structure. Milks from both species are exceptionally complex fluids containing many systems. To understand the nature of milk it is helpful to classify the components according to their size and concentration with solubility in milk, or the lack thereof. These considerations lead to the concept of compartmentation as presented by Jensen et al. (39), and Ruegg and Blanc (60) for human milk, and by Walstra and Jenness (68) for bovine milk. Compartmentation is important. because it is one of the factors - along with enzyme activity and state of the intestinal absorptive surface - that control the flow of nutrients into the enterocyte when milk has been consumed. Table 2 contains data on the comparunents, some characteristics, and major constituents of bovine and human milks (3, 39, 60, 68). The contents, number, and sizes of constituents are for mature milk and are approximate. Note the wide range in sizes and contents of components. The interactions occurring among the components in the compartJournal of Dairy Science Vol. 73.
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ments contribute to the complexity. Discussion will be limited to the lipids and associated compounds in the milk fat globule membrane (MFGM), the globule, and the emulsion. THE MILK FAT GLOBULE AND MEMBRANE
Most of the lipids (98% or more) in both milks are TG and are located with traces of other nonpolar lipids in the globules. The globules are formed throughout the mammary epithelial cell, grow in size as they more toward the apical cell membrane, and are extruded into the alveolar lumen (34, 36, 42, 60). During the extrusion process, the globule is enveloped by portions of the cell membrane, which becomes the MFGM. The MFGM stabilizes the globules in an oil and water emulsion. Because the MFGM are derived from the same source in each species, the plasma membrane of the secreting cells, their compositions do not differ greatly (8, 42, 60). Buchheim et aI. (10) observed the surface of milk fat globules by freeze-etch electron microscopy. They found an array of filaments, .5 ~m in length on the human, but not the bovine, globules. The filaments. which were identified as glycoproteins, were removed by heating. The authors speculated that the filaments could serve to bind globules to intestinal mucosal cells or as receptors for lipases involved in fat digestion. Whatever their function, the presence of the filaments is a major difference in MFGM topography between the species. The membranes contain milk and cellular proteins, phospholipids, sterols, enzymes, macrominerals, microminerals, and other bipolar substances that are more or less firmly bound (34, 42). The lipids of bovine and human MFGM have been studied by Bracco et al.
225
MILK LIPIDS TABLE 2. Compartments and their constituents in mature bovine and human milks. l Major constituents Content Compartment description
Bovine
Human
(B)
(H)
Name
Content (%)
--(%)--
Aqueous phase True solution. I urn Whey proteins. 3 to 9 urn
Colloidal dispersion 11 to 55 urn, 10 I 6/dl Emulsion fat globules 4 ~, 1.1 x IO IO/dl fat globule membrane Absorbed layer Cells 8to40vm 104 to I (}'/dl
87.3
87.6
2.6 3.7
4.0
1. Compounds of Ca, Mg, P04 Na, K, Cl. C02, citrate, casein 2. Whey proteins: «-lactalbumin. lactoferrin. 19A, lysozyme, and serum albumin B, 20% of total N; H. 70% 3. Lactose and oligosaccharides: 4.8 and .1 B; 7.0 and 1.0% H 4. Nonprotein nitrogen compounds: glycocyamine, urea, amino acids. B, 5% of total N; H, 25% 5. Miscellaneous: B vitamins, ascorbic acid 6. Caseins: ~- and K-, « for B, Ca, P04 7. Fat globules: triacylglycerols. fat-soluble vitamins 8. Milk fat globule membrane: proteins, phospholipids, cholesterol, enzymes. trace minerals 9. Macrophages, neutrophils, lymphocytes. epithelial cells. leukocytes
1All figures are approximate. Compiled from (3, 39, 60.
(8), who published the only detailed comparison of the MFGM. This infonnation is given in Table 3. Compared with the globule. the membranes contained much more polar lipids, as would be expected. In an oil-water emulsion, bipolar substances will always locate at the interface. where they stabilize the emulsion by preventing coalescence of the globules. With the MFGM, the bipolar lipids are phospholipids, the free cholesterol in the unsaponifiable fractions and the products of lipolysis. FFA, diacylglycerols (00). and monoacylglycerols (MG). The MG, FFA, and soluble soaps of the latter are bipolar and would locate in the membrane where they could alter the surface topography. This effect has not been studied. It is known that the membrane of globules in homogenized milks contains mostly casein with much of the original MFGM translocated to the serum (42, 51). Beyond this, very few data exist on the composition of the membranes in homogenized bovine milk and infant fonnula. Because these membranes partially control the availability of nutrients for absorption in tenns of sequence of events, the data are important.
.7 as ash, B .2 as a'ih, H .6 B, H
4.9 B 8.0 H 30 mg N/dl B 50 mg N/dl H 2.6 B .3 H 3.7 B 4.0 H 2% of total lipid
68).
There are more PUFA in the MFGM than in the globules because of the relatively large content of phospholipids. However, the contribution to the total PUFA content is small because the phospholipids are only 1% or less of the total lipids. The core of the globule contains nonpolar substances; TG. cholesterol esters, retinyl esters, etc. The layering of TG with those of smaller molecular weight at or near the surface of the interface of the globule has been proposed, but was not thought to occur (42, 51, 60). However, Timmen and Patton (65) have observed differences in the fatty acid composition of small as compared with large globules in bovine milk. They separated whole milk into skim milk (small globules) and cream (large globules) and detennined the fatty acid composition of the TG from these fractions. Their data are presented in Table 4. Mean diameters of the globules in skim milk were always smaller than those in cream. Also. there were less 4:0 to 10: o and 18:0 and more 18:1 in the small than in the large globules. Also, these relationships changed in the globules from the underfed cow. Journal of Dairy Science Vol. 73.
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TABLE 3. Composition of human and bovine milk fat globules and membranes (percent of total lipids).} Human
Bovine
Lipid compounds
Fat globule
Fat globule membrane
Free fatly acids Monoacylglycerols Diacylglycerols Triacylglycerols Phospholipids Unsaponifiable
.4 Trace .7 98.1 .26 .31
7.3 .6 8.1 58.2 23.4 .96
1Adapted
Fat globule .3
Fat globule membrane 6.7 Trace 8.9 61.7 22.1 .89
98.6 .26 .28
from (8).
The small globules represented only 5% of the total TO. Timmen and Patton (65) suggest that the differences are consistent with alternative use of short- chain acids or 18: 1 converted from 18:0 to maintain liquidity at body temperature of milk fat globules and their precursors. intracellular lipid droplets. Similar data are not available for human milk fat globules. One of the procedural difficulties in isolation of MFOM is the loss of loosely bound components during TO processing (29. 42). The procedures have been rigorous, often involving separation of the cream and churning. Patton and Huston (54) have devised a method for the isolation of globules which minimizes
loss of membrane components and applied it to human, bovine, and caprine milks. Their procedure should be used when possible. although it may not be applicable to large quantities of milk. The bovine MFOM has been studied because of its role in emulsion stability and both membranes because of their involvement in the globule extrusion process. The equally important function of rate control during absorption of nutrients through a time frame has received almost no attention. An example of rate of sequence control is the intestinal absorption of retinyl esters from a milk fat globule. These compounds are buried in the globule. The esters
TABLE 4. Differences between fal globules of creams and corresponding skim milks in triacylglycerol fatty acid composition for milks produced under various conditions.}
Item
Trial 1, herd Pasture
Trial 2, Individual Pasture
Trial 3. Herd Bam
Globule size 2 Cream Skim milk
2.81 1.41
2.93 1.02
3.17 1.77
-.48 -.02 +.02 +.95 -.98 +.06 +.19
-.48 +.06 +.26 -.09 -.78 +.53 +.07
Fatty acids4 4:0 to 10:0 12:0 14:0 16:0 18:0 18:1 18:2
-.21 .01 -1.07 -.98 +.78 +.66
Trial 4. Individual Bam ND 3 ND (g/IOO g fat) -.79 -.10 +.78 -.89 +.97 -.15
Trial 5, Individual Bam
Trial 6. Individual Underfed
2.76 1.50
3.33 1.35
-1.38 -.43 -.41 +.10 -.60 +1.24 +.22
+.12 +.19 +.59 +.99 -.48 -1.22 -.02
tDifferences are composition of triacylglycerol fatty acids from skim milk less those from cream, reference (65). 2Arithmetic mean of globule size diameters in micrometers (do). 3ND = Not determined. 4Designated by carbon chain length and number of double bonds. Journal of Dairy Science Vol. 73.
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TABLE 5. [mponant parameters of the fat globule dispersion in human colostrum and mature human and bovine milk. l
Range
Average Mature
Parameter
Human colostrwn
Fat content. %. wt/vol Globules in milk. noJml Surface area of fat in milk. m 2/ml Surface area of I g of fat. m 2 Average diameter. ~
2.6 6 x 10 10 -fyn 3.3 1.5
3.3
human milk 1.1 x 10 10
.054 1.4 4.0
Bovine milk 3.7-4.1 1.5 x 10 10 .06-.12 1.4-2.9 2.5-4.6
Homogenized bovine milk 3.7-4.1
12 10_10 14 4-1.2 10-30 .2-7
1Adapted from (59).
must be made available through the membrane to the appropriate esterases before they can be absorbed into the enterocyte, and this will occur at some point after the globules enter the small intestine. Here were see both rate and sequence control. THE EMULSION
Information on the dispersion of fat globules in human colostrum and mature human and cow's milk is presented in Table 5 (59). Although we have given average diameters for fat globules in Tables 2 and 5, these values are not representative of the size distributions. Curves of the distributions reveal subpopularions of different sized globules. Small globules with a size frequency maximum of about I J.lm comprise 70 to 90% of the total but only a small part of the total fat weight. The 4-J.lm globules contain the largest amount of fat but only 10 to 30% of the numbers. Large globules of 8 to 12 J.l.m are only .0 I% of the numbers but contain I to 4% of the fat. Note the changes that occurred in human milk as lactation progressed. The fat content increased, but the number of globules and the surface area decreased. Homogenization greatly increases the number of globules and their surface area. During this process, the smaller globules are recoated primarily with casein (42,51). Many of the constituents of the original membrane: phospholipids, etc. transfer into the milk serum (29, 42, 51). MILK LIPIDS
The average compositions for mature milks are shown in Table 6 (4, 12, 34, 36, 55). The overwhelming mass of the lipids is TG with much smaller quantities of sterols and phospho-
lipids, which are primarily associated with the membrane. The sterols are mostly cholesterol with about 15% of this in the ester form. Traces of hydrocarbons-carotenoids, retinyl esters. squalene, etc.-are found. ]0 freshly drawn and extracted milk, only traces of FFA, DG, and MG will be detected. The presence of large quantities of these and smaller amounts of TG is indicative of lipolysis. Both milks should be extracted immediately. frozen to -700C, or pasteurized to prevent lipolytic action (5, 34, 35). Lipolysis will not change the total fatty acid composition but will alter the relative amounts of FFA, TG, DG, and MG. The effects of lipolysis can be striking. For examples. see Table 3 where the FFA. DG. and MG are associated with the MFGM lipids. The large preponderance of TG makes it difficult to quantitate or resolve the other lipids. Bitman et a1. (4) separated the polar and nonpolar lipids of human milk with a Sep-Pak col-
TABLE 6. Composition (%) of mature bovine and human milk lipids. Lipids
Bovine 1
Hydrocarbons Sterol esters Triacylglycerols Diacylgl)'cerols Monoaeylglycerol Free fally acids Sterols3 Phospholipids
Trace Trace 98+ Trace Trace Trace
Trace Trace 98+ Trace Trace Trace
.3
.4 13
.8
IAdapted from (12. 36. 54). 2Adapled from (4. 34). 3About 10 to 20 mg/dL Journal of Dairy Science Vol. 73.
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JENSEN ET AL.
urnn (Waters Associates, Inc., Milford. MA), while the dry column procedure has been employed for exlraction and separation of bovine (49) and human milk lipids (16. 25). Bitman et aI. resolved the nonpolar (4) and polar lipids (6) of human milk into classes by TLC. then quantitated them with densitometry. Hundrieser et a1. (26) developed a method for the measurement of phospholipid phosphorous in human milk which does not require acid digestion. Later, an HPLC procedure for separation of the phospholipids was devised (27). The best procedure for routine determination of cholesterol is that of Bachman et aI. (1). With this technique. the milk is saponified directly. extracted with hexane, and the cholesterol in the eXlract determined by treatment with o-phthalaldehyde and spectrophotometry. Gas-liquid chromatography should be employed if the investigator is searching for sterols other than cholesterol. e.g., desmosterol in human milk (15). High performance liquid chromatography can also be used for human (23) and bovine milk sterols (28). Methods have been detailed for the analysis of these lipids in human milk (33. 34, 35) and in bovine milk (13. 36).
TRIACYLGLYCEROLS
The composition of TG is usually defined in terms of the kinds and amounts of fatty acids present and will be discussed later. In this section, we add the dimension of structure to the definition. Structure is important because it is in the form of esters as TG that milk lipids are presented to lipases and that bovine milk lipids are processed. Structure includes the distribution of fatty acids within the TG molecule and among the TG molecules as well as the identification of the individual molecular species of TG (34. 36). The lipids in both milks contain about 10 major fatty acids (see Table 7) (11); therefore, it would be theoretically possible to have 1 x loJ or 1000 TG species if all the acids were randomly distributed. The total theoretical possibilities are much greater, because bovine milk lipids contain almost 400 fatty acids and human milk lipids contain nearly 200. With 400 fany acids the theoretical maximum is 400 x 103, or 64 million TG species! Although the quantities of the major individual molecular TG species can be determined in a fat. this has not been done for either of the
TABLE 7. Positional distribution of fatly acids in the sn triacylglycerols (TO) from human milk and normal and linoleic acid-rich bovine milk. 1
Fatty acids 2
Human milk 3 TO
50-1
50-2
Linoleic acid-rich cow's milk4
Cow's milk sn-3
TG
50-1
50-2
sn-3
TG
1.4 1.9 4.9 9.7 2.0 34.0 2.8 1.3 10.3 30.0 1.7
.9 .7 3.0 6.2 17.5 2.9 32.3 3.6 1.0 9.5 18.9 3.5
35.4 12.9 3.6 6.2 .6 6.4 1.4 5.4 1.4 .1 1.2 23.1 2.3
10.8 3.8 1.5 2.7 3.4 7.5 1.0 15.7 .8 .4 10.4 26.9 15.3 Trace
sn-l
sn-2
50-3
(moVl00 mol) 4:0 6:0 8:0 10:0 12:0 14:0 15:0 16:0 16:1 17:0 18:0 18:1 18:2 18:3
2.9 7.3 9.4 .8 27.0 3.6
1.1 4,5 6.5 .6 18.7 3.4
1.6 6.9 15.4 .9 57.1 1.6
5.9 10.4 6.4 1.0 5.3 5.8
7.1 34.2 7.9 Trace
14.2 44.0 7.2
4.9 8.1 3.7
2.2 50.5 12.7
11.8 4.6 1.9 3.7 3.9 11.2 2.1 23.9 2.6 .8 7.0 24.0 2.5 Trace
1Adapted from (11).
2Designated by carbon chain length and number of double bonds. 3Mean of two samples. 4Mean of four samples. Journal of Dairy Science Vol. 73.
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1.6 2.4 3.3 8.3 1.2 22.1 .8 .6 14.3 32.3 13.1
.6 .7 3.0 4.8 12.4 1.4 23.3 1.2 .4
11.1 24.4 16.8
32.3 10.6 2.3 2.7 2.0
1.7 .4 1.7 .5 .1 5.7 24.1 16.0
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MILK LIPIDS
milk TG, but it should be, because the structure of the TG influences the action of lipolytic enzymes and. therefore, absorption. In human milk TG, location of 16:0 in the sn-2 position enhances absorption of dietary fats (66). This information could also assist in the design of milk fat for infant formulas that is more like human milk. Structure of bovine milk TG is responsible for the melting points, crystallization behavior, and rheological properties of bovine milk fat as globules and in butter and butter oil. The fatty acid composition and, hence, bovine milk TG structure is not greatly affected directly by ordinary changes in diet because of the biohydrogenation and production of short-chain fatty acids in the rumen. Nevertheless, changes can occur, e.g., the effect of underfeeding a cow as presented in Table 4 [see also (2)]. The fatty acid composition of bovine milk can be changed markedly by feeding protected oils rich in 18:2. The oils are encapsulated in denatured casein. When fed to the cow, the capsules pass through the rumen unaffected by hydrogenation and the casein is digested in the abomasum releasing the oils. A milk rich in 18:2 results (Table 7). Note the five- to sixfold increase in total 18:2 and the relatively equal distribution of the acid. Because the fatty acid composition of human milk TG responds to changes in diet, the structure of the TG would also be expected to change. We have shown that when a diet high in polyunsaturated food oils is consumed, 18:2 displaces 16:0 from the sn-2 position (37). Alteration in diet changes the TG structure of both milks. The distribution of fatty acids in human and bovine milk TG is asymmetrical (Table 7). The presence of enantiomers is indicated where there are major differences in the compositions of the sn-l and sn-3 positions. In these samples of human milk TG, the positional distribution resembles that of unrearranged lard in which a substantial part of the 16:0 is also located in the sn-2 position (11). In the bovine sample, all of the 4:0, 93% of the 6:0, and 63% of the 8:0 are esterified to the sn-3 position. With additional analyses, such as determination of the relative molecular weights (carbon numbers) of the TG by GLC, several individual TG in the milks have been tentatively identified. These are: bovine milk (34): rac-14: 0-18:0-18:1, rae- 14:0-18:1-18:0 or rae-18: 0-14:0-18:1, sn- 1,2 (18:0-18:1, 16:0, 4:0, 6:0, or 8:0; and human milk (38): sn-18:0-16:0-14:
0, sn-18:0-l6:O-l6:0, sn-18:1-l6:O-l8:1, sn-18: 1-16:0-18:2, and sn-18:1-18:l-18:2. Obviously, the structures are distinctive. Most of the fatty acids in human milk lipids will eventually, enter the enterocytes via the micellar pathway. The fatty acids up through C12; human, 10.2 mol/lOO mol and bovine, 25.9 mol/loo mol (Table 7), will pass into the portal vein, some through the stomach wall, and the remainder into the enterocyte independent of the micellar pathway. These shorter acids are then carried to the liver where they are quickly oxidized, providing energy. The metabolism of bovine milk TG is unique since no other dietary lipid consumed in any quantity is divided to the extent above, Le., about 25 moll 100 mol of the fatty acids bypassing the micellar pathway of intestinal absorption. This aspect of lipid metabolism has not been investigated. In both milks, the emulsified globules with large surface area are an ideal substrate for lipases (9, 59). Most fluid bovine milk is homogenized, which increases the number of globules and the surface area, providing more space for enzyme activity. In fact, the high coefficients of absorption of lipids in both milks are due in part to the vast surface area of emulsified globules (Table 5). The lipids in infant formulas are also homogenized. The effects of this treatment should be considered when comparative absorptions are studied, but this has not been done. The other factors controlling absorbability are compartmentation in the milks, condition of the intestinal surface, and activities of the lipases and related compounds. We will not discuss the intestinal surface. The TG in both milks are hydrolyzed by gastric and lingual (20) and pancreatic lipases (67). In human milk, the bile salt-stimulated lipase (BSSL) is very active in the breastfed infant's intestine (52). The globule and membrane then have profound effects. In addition to the items mentioned, they carry cholesterol, fat-soluble vitamins, flavor compounds, and other materials soluble in fat. In summary, the globule and membrane are vehicles and compartments, carrying and providing milk components as needed in timed sequences. PHOSPHOLIPIDS
The composition of the milk phospholipids are listed in Table 8 (6, 34, 36, 45. 46, 55). As Jownal of Dairy Science Vol. 73,
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JENSEN ET AL.
mentioned earlier, these originated from the membranes of the mammary cells and in fresh quiescent milk will be found mostly in the MFGM. Depending upon the severity of the treatment, more or less material will relocate into the skim milk. In addition to their functions as emulsifiers, and MFGM stabilizers, the phospholipids are a source of long-chain PUFA. They bind cations, and possibly interact with digestive enzymes. Because of their location and PUFA content, they are foci of autoxidation. The gangliosides inhibit enterotoxins from Vibrio cholerae and Escherichia coli even though they are present in very small quantities (45). These lipids are one of the many host defense mechanisms in human milk and explain, in part, why in some breastfed infants, the incidence and severity of diarrhea is less than in their nonbreast fed peers. The quantity of active ganglioside, GMt, is much greater in human (12 Ilg/L) than in bovine (1 Ilg/L) milk. The other microphospholipids may also have. as yet, undiscovered nonnutritive functions. STEROLS
The major sterol in both milks is cholesterol in amounts ranging from 10 to 20 mg/dl (34, 36) with the quantities related to the fat content. Trace amounts of other sterols are present, e.g., lanosterol in bovine milk and desmosterol
TABLE 8. Compositions of phospholipids in bovine and human milk lipids. Phospholipid Phosphatidy lcholine Phosphatidy lethanolamine Phosphatidy lserine Phosphatidylinosiliol Sphingomyelin Lysophosphalidy lcholine Lysophosphatidylethanolamine Plasmalogens Diphosphalidylglycerol Ceramides Cerebrosides Gangliosides. mg/L
- (g/IOO g phospholipid) 33.8 27.5 36.3 19.9 3.9 8.4 5.3 5.2 20.8 38.9 Trace Trace Trace
Trace
3.0 Trace Trace Trace 11 3
Trace Trace 11 3
tFrom (34, 36, 46, 55). 2From (6). 3From (45). Journal of Dairy Science Vol. 73.
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(15) and some phytosterols (23) in human milk. Whole bovine milk is obviously not a high cholesterol food, containing at the most about 200 mg/L. It is unfortunate that the importance of bovine milk as a source of B vitamins, vitamin A, calcium, and high quality protein has been overshadowed by "cholesterolphobia". Recent work by McNamara et a1. (50) indicated that dietary cholesterol had little influence on the plasma cholesterol of their subjects over a 6-wk period. In 69% of the subjects, increased cholesterol intake was accompanied by a decrease in cholesterol fractional absorption or endogenous cholesterol synthesis. The 50 male subjects were asymptomatic for cardiovascular disease and free of secondary causes of hyperlipidemia. Diets rich in saturated fatty acids, (polyunsaturated (P); saturated (S) ratio of .3) as compared with one high in polyunsaturated fatty acids (P:S ratio of 1.5), resulted in higher plasma cholesterol levels. It appears that the impact of diet on plasma cholesterol is individualized; saturated fatty acids are more important than cholesterol in most of the subjects studied. We will discuss the effects of fatty acids on plasma cholesterol levels later. However, in only 30% of the small, healthy group of male subjects studied, endogenous cholesterol synthesis was not inhibited by dietary cholesterol. The type of dietary fat had a large and more consistent effect on plasma cholesterol than dietary cholesterol. Therefore, the fatty acid composition of dietary fats must be considered. Based on the small number of healthy subjects in this study, it would appear that only about 30% of the population would need to severely limit dietary cholesterol. However, the amount and kind of dietary fat still needs attention [See (44, 62) for reviews]. FATTY ACIDS
The fatty acids in both milks have been analyzed extensively with GLC. Analysis of bovine milk fatty acids is difficult because of problems concerning loss and separation of the short-chain fatty acid esters. These have been overcome by the use of temperature programming of the instrument (31, 40) and conversion of the fatty acids to butyl instead of methyl esters (13, 31. 40, 53). Excellent resolution of many fatty acids, including trans isomers, can
231
MILK LIPIDS
TABLE 9. Fatty acid composition of bovine and human milks.
Fany acid'
Bovine2
- - - (g/I00 g fat) - - 3.3 4:0 Trace 6:0 I~ Trace 8:0 1.3 10:0 3.0 1.3 12:0 3.\ 3.1 14.2 5.1 \4:0 \5:0 1.3 Trace to .4 16:0 42.7 20.2 16:1 3.7 5.7 6.0 18:0 5.7 46.4 18:1 16.7 13.0 18:2 1.6 1.4 18:3 1.8 36.1 Tow saturates 76.2 28.8 Hypercholesterolemic4 61.3 1Designated by carbon chain length and number of double bonds. 2From (36). 3From (34).
~otaI of hypercholesterolemic fatty acids (12:0 + 14:0 + 15:0 + 16:0).
be obtained with regular capillary or wide bore capillary columns. These can and should be used routinely. The compositions of the major fatty acids, determined with packed columns, are given in Table 9 (34. 36). Note that as compared with bovine milk, human milk fatty acids have little 4:0 to 8:0, less saturated fatty acids, and more 18:1 and 18:2. Human milk
also contains more long-chain PUFA, which are not listed here. It has been customary to report fatty acid compositions in weight percent, (g of fatty acid/UK) g fat) or normalized data. This is satisfactory when only the fatty acid composition is needed, but if any type of intervention is done that produces a real change in one or more acids, the relative weight percents of the others must change artificially to total 100%. These changes could be misleading because they are not real. Intervention studies should be reported as gravimetric data (mg of fatty acid/ 100 ml milk) (48). Gravimetric data can be determined by use of an internal standard during GLe analysis or by calculation from the fat content. Gravimetric data are a must for the nutritionist. See Table 10 for a comparison of the methods (22). On d 1 of lactation, the sample contained 739.9 mg of 18:1/100 ml or 38.7% of the total fat. At d 30, the amount was 1267.7 mg, but the weight percent had decreased to 35. It is very important to use gravimetric data for human milk, because individual samples are almost always analyzed, and the fatty acid composition responds to dietary changes. It is also important to know the volume of milk involved. Unless an intervention study is being planned which might affect the fatty acid profile and where gravimetric data should be used, the method of reporting is not a problem with bovine milk fatty acids. This is because most market milk has been extensively pooled and will have about the same fat content.
TABLE 10. The fatty acid com~ition of human milk as mg of fatty acids per 100 mI milk (gravimetric)1 and g of fany acid per 100 g fat (normalized).2 Day of lactation
~~ 12:0 14:0 16:0 18:0 18:1 18:2 22:603
8
I
13.4 1 65.8 455.8 179.8 739.9 184.5 4.2
1.1 2
4.3 26.2 9.3 38.7 9.7 .2
93.8 1 163.3 769.3 294.3 1244.0 365.1 8.7
3.~ 5.9 24.8 8.6 36.4 10.8 .3
30 140.5' 210.7 758.9 305.1 1267.7 423.4 5.7
5.5 2 7.2 23.1 8.4 35.0 11.8 .2
lMilligrams of fatty acid per 100 ml milk. Adaple
Lipids of Bovine and Human Milks: A Comparison 1 ROBERT G. JENSEN, ANN M. FERRIS, CAROL J. LAMMI-KEEFE, and ROBIN A. HENDERSON Department of Nutritional Sciences University of Connecticut Storrs 06269-4017 ABSTRACT
ing. In our first review on human milk lipids, we observed that sampling had been poorly done and that modern methods of analysis had not always been used (41). Fortunately, this has changed and now there are reliable data on the c~mposition of human milk. In this paper, we wIll compare data on the lipids in bovine and human milks. Blanc (3) and Renner (58) have presented reviews on the comparison. Other reviews that we have used are by Christie (12, 13) and Jensen and Clark (36) on bovine milk lipids and by Jensen (38) on human milk lipids. A short version of this review has been presented (38). This paper is not intended to be comprehensive.
Human and bovine milks contain about 3 to 5% total lipid, existing as emulsified globules 2 to 4 Jlm in diameter and coated with a membrane derived from the secreting cell. About 98% or more of the lipid is triacylglycerol, which is found in the globule. Phospholipids are about .5 to 1% of total lipids and sterols are .2 to .5%; these are mostly located in the globule membrane. Cholesterol is the major sterol. The major differences are in fatty acid composition, triacylglycerol structure, and the response of fatty acids in human milk to changes in diet. Bovine milk contains substantial quantities of 4:0 to 10:0, about 2% 18:2, and almost no other long-chain polyunsaturated fatty acids. The fatty acid composition is not altered by ordinary changes in diet. Human milk contains very little 4:0 to 10:0, 10 to 14% 18:2, and small quantities of other polyunsaturates. The triacylglycerol structure differs, with much of the sn-2 position occupied by 16:0 in human milk and 4:0 to 10:0 at sn-3 in bovine milk. The effects of milk cholesterol and fatty acids on human blood cholesterol levels are discussed. (Key words: lipids in milk, cholesterol)
THE NATURE OF MILKS
INTRODUCTION
Lipids of human milk have not been investigated as extensively as those of bovine milk. However, this is changing, because research on bovine milk lipids is almost at a standstill, but investigations of human milk lipids are inereas-
Received June 22. 1989. Accepted August 18. 1989. lScienti~C Contri~ution Nwnber 1295. StOrTS Agricultural Ellperunent Stauon, The University of Connecticut. StOrTS 06269-40 17. 1990 ] Dairy Sci 73:223-240
We have listed in Table 1, (3, 32, 46) the general composition of human and bovine milks. The data in Table 1 represent an oversimplification of the complexity of milk, but note that human milk contains less protein and ~ore lactose than bovine milk. More profound dIfferences include the higher nonprotein nitrogen content of human milk. The figures are averages for human milk, which change as lactation progresses. Bovine milk is consistently uniform because the milk pooled for processing is usually homogenized and colostrum is excluded. With human milk, each sample from an individual at a particular time can differ in content and composition from previous samples or from those of other women otherwise matched for time, age, diet, etc. (32, 36, 41). Almost always the infant receives an individual feeding of unprocessed human milk, which is unique in itself. Compared with bovine milk, volumes of human milk are small, up to 100 mllfeeding with an average of about 750 mIld for mature milk (34). Many factors can affect the volume and composition of human milk, the most influential being time postpartum (stage of lactation) and diet. These influences are essentially elimi-
223
224
JENSEN ET AL.
TABLE 1. General composition (%) of human and bovine milks. t Casein % of
Milk
Protein
protein
Fat
Lactose
TOlal solids
Ash
Kilocalories
12.1
.2
12.7
.7
72 58 75
(%)
Human Mature. 36 d Colostrum, 3 d Bovine
1.0 2.3 3.4
40 82
3.9 3.0 3.7
6.8 5.5 4.8
1Adapled from (3. 32. 46).
nated in bovine milk by pooling. In Table I, the protein content of human colostrum is higher than in mature milk, 2.3 vs. 1.0%, and the fat and lactose lower, 3.0 vs. 3.9% and 5.5 vs. 6.8%. The volume of colostrum is also lower. The biosynthetic pathways for bovine and human milk lipids are essentially identical. The major exceptions are: 1) bovine rumen activity, which provides short-chain fany acids, 4:0 to 10:0, biohydrogenates dietary polyunsaturated fatty acids (PUFA), decreasing the amounts of these in milk and producing trans and positional isomers of unsaturates, and limiting the effects of dietary changes on the composition of bovine milk fatty acids; and 2) differences in acylglycerol transferases, which result in species specific patterns of triacylglycerol (TG) structure. Milks from both species are exceptionally complex fluids containing many systems. To understand the nature of milk it is helpful to classify the components according to their size and concentration with solubility in milk, or the lack thereof. These considerations lead to the concept of compartmentation as presented by Jensen et al. (39), and Ruegg and Blanc (60) for human milk, and by Walstra and Jenness (68) for bovine milk. Compartmentation is important. because it is one of the factors - along with enzyme activity and state of the intestinal absorptive surface - that control the flow of nutrients into the enterocyte when milk has been consumed. Table 2 contains data on the comparunents, some characteristics, and major constituents of bovine and human milks (3, 39, 60, 68). The contents, number, and sizes of constituents are for mature milk and are approximate. Note the wide range in sizes and contents of components. The interactions occurring among the components in the compartJournal of Dairy Science Vol. 73.
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ments contribute to the complexity. Discussion will be limited to the lipids and associated compounds in the milk fat globule membrane (MFGM), the globule, and the emulsion. THE MILK FAT GLOBULE AND MEMBRANE
Most of the lipids (98% or more) in both milks are TG and are located with traces of other nonpolar lipids in the globules. The globules are formed throughout the mammary epithelial cell, grow in size as they more toward the apical cell membrane, and are extruded into the alveolar lumen (34, 36, 42, 60). During the extrusion process, the globule is enveloped by portions of the cell membrane, which becomes the MFGM. The MFGM stabilizes the globules in an oil and water emulsion. Because the MFGM are derived from the same source in each species, the plasma membrane of the secreting cells, their compositions do not differ greatly (8, 42, 60). Buchheim et aI. (10) observed the surface of milk fat globules by freeze-etch electron microscopy. They found an array of filaments, .5 ~m in length on the human, but not the bovine, globules. The filaments. which were identified as glycoproteins, were removed by heating. The authors speculated that the filaments could serve to bind globules to intestinal mucosal cells or as receptors for lipases involved in fat digestion. Whatever their function, the presence of the filaments is a major difference in MFGM topography between the species. The membranes contain milk and cellular proteins, phospholipids, sterols, enzymes, macrominerals, microminerals, and other bipolar substances that are more or less firmly bound (34, 42). The lipids of bovine and human MFGM have been studied by Bracco et al.
225
MILK LIPIDS TABLE 2. Compartments and their constituents in mature bovine and human milks. l Major constituents Content Compartment description
Bovine
Human
(B)
(H)
Name
Content (%)
--(%)--
Aqueous phase True solution. I urn Whey proteins. 3 to 9 urn
Colloidal dispersion 11 to 55 urn, 10 I 6/dl Emulsion fat globules 4 ~, 1.1 x IO IO/dl fat globule membrane Absorbed layer Cells 8to40vm 104 to I (}'/dl
87.3
87.6
2.6 3.7
4.0
1. Compounds of Ca, Mg, P04 Na, K, Cl. C02, citrate, casein 2. Whey proteins: «-lactalbumin. lactoferrin. 19A, lysozyme, and serum albumin B, 20% of total N; H. 70% 3. Lactose and oligosaccharides: 4.8 and .1 B; 7.0 and 1.0% H 4. Nonprotein nitrogen compounds: glycocyamine, urea, amino acids. B, 5% of total N; H, 25% 5. Miscellaneous: B vitamins, ascorbic acid 6. Caseins: ~- and K-, « for B, Ca, P04 7. Fat globules: triacylglycerols. fat-soluble vitamins 8. Milk fat globule membrane: proteins, phospholipids, cholesterol, enzymes. trace minerals 9. Macrophages, neutrophils, lymphocytes. epithelial cells. leukocytes
1All figures are approximate. Compiled from (3, 39, 60.
(8), who published the only detailed comparison of the MFGM. This infonnation is given in Table 3. Compared with the globule. the membranes contained much more polar lipids, as would be expected. In an oil-water emulsion, bipolar substances will always locate at the interface. where they stabilize the emulsion by preventing coalescence of the globules. With the MFGM, the bipolar lipids are phospholipids, the free cholesterol in the unsaponifiable fractions and the products of lipolysis. FFA, diacylglycerols (00). and monoacylglycerols (MG). The MG, FFA, and soluble soaps of the latter are bipolar and would locate in the membrane where they could alter the surface topography. This effect has not been studied. It is known that the membrane of globules in homogenized milks contains mostly casein with much of the original MFGM translocated to the serum (42, 51). Beyond this, very few data exist on the composition of the membranes in homogenized bovine milk and infant fonnula. Because these membranes partially control the availability of nutrients for absorption in tenns of sequence of events, the data are important.
.7 as ash, B .2 as a'ih, H .6 B, H
4.9 B 8.0 H 30 mg N/dl B 50 mg N/dl H 2.6 B .3 H 3.7 B 4.0 H 2% of total lipid
68).
There are more PUFA in the MFGM than in the globules because of the relatively large content of phospholipids. However, the contribution to the total PUFA content is small because the phospholipids are only 1% or less of the total lipids. The core of the globule contains nonpolar substances; TG. cholesterol esters, retinyl esters, etc. The layering of TG with those of smaller molecular weight at or near the surface of the interface of the globule has been proposed, but was not thought to occur (42, 51, 60). However, Timmen and Patton (65) have observed differences in the fatty acid composition of small as compared with large globules in bovine milk. They separated whole milk into skim milk (small globules) and cream (large globules) and detennined the fatty acid composition of the TG from these fractions. Their data are presented in Table 4. Mean diameters of the globules in skim milk were always smaller than those in cream. Also. there were less 4:0 to 10: o and 18:0 and more 18:1 in the small than in the large globules. Also, these relationships changed in the globules from the underfed cow. Journal of Dairy Science Vol. 73.
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JENSEN ET AL.
TABLE 3. Composition of human and bovine milk fat globules and membranes (percent of total lipids).} Human
Bovine
Lipid compounds
Fat globule
Fat globule membrane
Free fatly acids Monoacylglycerols Diacylglycerols Triacylglycerols Phospholipids Unsaponifiable
.4 Trace .7 98.1 .26 .31
7.3 .6 8.1 58.2 23.4 .96
1Adapted
Fat globule .3
Fat globule membrane 6.7 Trace 8.9 61.7 22.1 .89
98.6 .26 .28
from (8).
The small globules represented only 5% of the total TO. Timmen and Patton (65) suggest that the differences are consistent with alternative use of short- chain acids or 18: 1 converted from 18:0 to maintain liquidity at body temperature of milk fat globules and their precursors. intracellular lipid droplets. Similar data are not available for human milk fat globules. One of the procedural difficulties in isolation of MFOM is the loss of loosely bound components during TO processing (29. 42). The procedures have been rigorous, often involving separation of the cream and churning. Patton and Huston (54) have devised a method for the isolation of globules which minimizes
loss of membrane components and applied it to human, bovine, and caprine milks. Their procedure should be used when possible. although it may not be applicable to large quantities of milk. The bovine MFOM has been studied because of its role in emulsion stability and both membranes because of their involvement in the globule extrusion process. The equally important function of rate control during absorption of nutrients through a time frame has received almost no attention. An example of rate of sequence control is the intestinal absorption of retinyl esters from a milk fat globule. These compounds are buried in the globule. The esters
TABLE 4. Differences between fal globules of creams and corresponding skim milks in triacylglycerol fatty acid composition for milks produced under various conditions.}
Item
Trial 1, herd Pasture
Trial 2, Individual Pasture
Trial 3. Herd Bam
Globule size 2 Cream Skim milk
2.81 1.41
2.93 1.02
3.17 1.77
-.48 -.02 +.02 +.95 -.98 +.06 +.19
-.48 +.06 +.26 -.09 -.78 +.53 +.07
Fatty acids4 4:0 to 10:0 12:0 14:0 16:0 18:0 18:1 18:2
-.21 .01 -1.07 -.98 +.78 +.66
Trial 4. Individual Bam ND 3 ND (g/IOO g fat) -.79 -.10 +.78 -.89 +.97 -.15
Trial 5, Individual Bam
Trial 6. Individual Underfed
2.76 1.50
3.33 1.35
-1.38 -.43 -.41 +.10 -.60 +1.24 +.22
+.12 +.19 +.59 +.99 -.48 -1.22 -.02
tDifferences are composition of triacylglycerol fatty acids from skim milk less those from cream, reference (65). 2Arithmetic mean of globule size diameters in micrometers (do). 3ND = Not determined. 4Designated by carbon chain length and number of double bonds. Journal of Dairy Science Vol. 73.
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MILK LIPIDS
TABLE 5. [mponant parameters of the fat globule dispersion in human colostrum and mature human and bovine milk. l
Range
Average Mature
Parameter
Human colostrwn
Fat content. %. wt/vol Globules in milk. noJml Surface area of fat in milk. m 2/ml Surface area of I g of fat. m 2 Average diameter. ~
2.6 6 x 10 10 -fyn 3.3 1.5
3.3
human milk 1.1 x 10 10
.054 1.4 4.0
Bovine milk 3.7-4.1 1.5 x 10 10 .06-.12 1.4-2.9 2.5-4.6
Homogenized bovine milk 3.7-4.1
12 10_10 14 4-1.2 10-30 .2-7
1Adapted from (59).
must be made available through the membrane to the appropriate esterases before they can be absorbed into the enterocyte, and this will occur at some point after the globules enter the small intestine. Here were see both rate and sequence control. THE EMULSION
Information on the dispersion of fat globules in human colostrum and mature human and cow's milk is presented in Table 5 (59). Although we have given average diameters for fat globules in Tables 2 and 5, these values are not representative of the size distributions. Curves of the distributions reveal subpopularions of different sized globules. Small globules with a size frequency maximum of about I J.lm comprise 70 to 90% of the total but only a small part of the total fat weight. The 4-J.lm globules contain the largest amount of fat but only 10 to 30% of the numbers. Large globules of 8 to 12 J.l.m are only .0 I% of the numbers but contain I to 4% of the fat. Note the changes that occurred in human milk as lactation progressed. The fat content increased, but the number of globules and the surface area decreased. Homogenization greatly increases the number of globules and their surface area. During this process, the smaller globules are recoated primarily with casein (42,51). Many of the constituents of the original membrane: phospholipids, etc. transfer into the milk serum (29, 42, 51). MILK LIPIDS
The average compositions for mature milks are shown in Table 6 (4, 12, 34, 36, 55). The overwhelming mass of the lipids is TG with much smaller quantities of sterols and phospho-
lipids, which are primarily associated with the membrane. The sterols are mostly cholesterol with about 15% of this in the ester form. Traces of hydrocarbons-carotenoids, retinyl esters. squalene, etc.-are found. ]0 freshly drawn and extracted milk, only traces of FFA, DG, and MG will be detected. The presence of large quantities of these and smaller amounts of TG is indicative of lipolysis. Both milks should be extracted immediately. frozen to -700C, or pasteurized to prevent lipolytic action (5, 34, 35). Lipolysis will not change the total fatty acid composition but will alter the relative amounts of FFA, TG, DG, and MG. The effects of lipolysis can be striking. For examples. see Table 3 where the FFA. DG. and MG are associated with the MFGM lipids. The large preponderance of TG makes it difficult to quantitate or resolve the other lipids. Bitman et a1. (4) separated the polar and nonpolar lipids of human milk with a Sep-Pak col-
TABLE 6. Composition (%) of mature bovine and human milk lipids. Lipids
Bovine 1
Hydrocarbons Sterol esters Triacylglycerols Diacylgl)'cerols Monoaeylglycerol Free fally acids Sterols3 Phospholipids
Trace Trace 98+ Trace Trace Trace
Trace Trace 98+ Trace Trace Trace
.3
.4 13
.8
IAdapted from (12. 36. 54). 2Adapled from (4. 34). 3About 10 to 20 mg/dL Journal of Dairy Science Vol. 73.
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JENSEN ET AL.
urnn (Waters Associates, Inc., Milford. MA), while the dry column procedure has been employed for exlraction and separation of bovine (49) and human milk lipids (16. 25). Bitman et aI. resolved the nonpolar (4) and polar lipids (6) of human milk into classes by TLC. then quantitated them with densitometry. Hundrieser et a1. (26) developed a method for the measurement of phospholipid phosphorous in human milk which does not require acid digestion. Later, an HPLC procedure for separation of the phospholipids was devised (27). The best procedure for routine determination of cholesterol is that of Bachman et aI. (1). With this technique. the milk is saponified directly. extracted with hexane, and the cholesterol in the eXlract determined by treatment with o-phthalaldehyde and spectrophotometry. Gas-liquid chromatography should be employed if the investigator is searching for sterols other than cholesterol. e.g., desmosterol in human milk (15). High performance liquid chromatography can also be used for human (23) and bovine milk sterols (28). Methods have been detailed for the analysis of these lipids in human milk (33. 34, 35) and in bovine milk (13. 36).
TRIACYLGLYCEROLS
The composition of TG is usually defined in terms of the kinds and amounts of fatty acids present and will be discussed later. In this section, we add the dimension of structure to the definition. Structure is important because it is in the form of esters as TG that milk lipids are presented to lipases and that bovine milk lipids are processed. Structure includes the distribution of fatty acids within the TG molecule and among the TG molecules as well as the identification of the individual molecular species of TG (34. 36). The lipids in both milks contain about 10 major fatty acids (see Table 7) (11); therefore, it would be theoretically possible to have 1 x loJ or 1000 TG species if all the acids were randomly distributed. The total theoretical possibilities are much greater, because bovine milk lipids contain almost 400 fatty acids and human milk lipids contain nearly 200. With 400 fany acids the theoretical maximum is 400 x 103, or 64 million TG species! Although the quantities of the major individual molecular TG species can be determined in a fat. this has not been done for either of the
TABLE 7. Positional distribution of fatly acids in the sn triacylglycerols (TO) from human milk and normal and linoleic acid-rich bovine milk. 1
Fatty acids 2
Human milk 3 TO
50-1
50-2
Linoleic acid-rich cow's milk4
Cow's milk sn-3
TG
50-1
50-2
sn-3
TG
1.4 1.9 4.9 9.7 2.0 34.0 2.8 1.3 10.3 30.0 1.7
.9 .7 3.0 6.2 17.5 2.9 32.3 3.6 1.0 9.5 18.9 3.5
35.4 12.9 3.6 6.2 .6 6.4 1.4 5.4 1.4 .1 1.2 23.1 2.3
10.8 3.8 1.5 2.7 3.4 7.5 1.0 15.7 .8 .4 10.4 26.9 15.3 Trace
sn-l
sn-2
50-3
(moVl00 mol) 4:0 6:0 8:0 10:0 12:0 14:0 15:0 16:0 16:1 17:0 18:0 18:1 18:2 18:3
2.9 7.3 9.4 .8 27.0 3.6
1.1 4,5 6.5 .6 18.7 3.4
1.6 6.9 15.4 .9 57.1 1.6
5.9 10.4 6.4 1.0 5.3 5.8
7.1 34.2 7.9 Trace
14.2 44.0 7.2
4.9 8.1 3.7
2.2 50.5 12.7
11.8 4.6 1.9 3.7 3.9 11.2 2.1 23.9 2.6 .8 7.0 24.0 2.5 Trace
1Adapted from (11).
2Designated by carbon chain length and number of double bonds. 3Mean of two samples. 4Mean of four samples. Journal of Dairy Science Vol. 73.
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1.6 2.4 3.3 8.3 1.2 22.1 .8 .6 14.3 32.3 13.1
.6 .7 3.0 4.8 12.4 1.4 23.3 1.2 .4
11.1 24.4 16.8
32.3 10.6 2.3 2.7 2.0
1.7 .4 1.7 .5 .1 5.7 24.1 16.0
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MILK LIPIDS
milk TG, but it should be, because the structure of the TG influences the action of lipolytic enzymes and. therefore, absorption. In human milk TG, location of 16:0 in the sn-2 position enhances absorption of dietary fats (66). This information could also assist in the design of milk fat for infant formulas that is more like human milk. Structure of bovine milk TG is responsible for the melting points, crystallization behavior, and rheological properties of bovine milk fat as globules and in butter and butter oil. The fatty acid composition and, hence, bovine milk TG structure is not greatly affected directly by ordinary changes in diet because of the biohydrogenation and production of short-chain fatty acids in the rumen. Nevertheless, changes can occur, e.g., the effect of underfeeding a cow as presented in Table 4 [see also (2)]. The fatty acid composition of bovine milk can be changed markedly by feeding protected oils rich in 18:2. The oils are encapsulated in denatured casein. When fed to the cow, the capsules pass through the rumen unaffected by hydrogenation and the casein is digested in the abomasum releasing the oils. A milk rich in 18:2 results (Table 7). Note the five- to sixfold increase in total 18:2 and the relatively equal distribution of the acid. Because the fatty acid composition of human milk TG responds to changes in diet, the structure of the TG would also be expected to change. We have shown that when a diet high in polyunsaturated food oils is consumed, 18:2 displaces 16:0 from the sn-2 position (37). Alteration in diet changes the TG structure of both milks. The distribution of fatty acids in human and bovine milk TG is asymmetrical (Table 7). The presence of enantiomers is indicated where there are major differences in the compositions of the sn-l and sn-3 positions. In these samples of human milk TG, the positional distribution resembles that of unrearranged lard in which a substantial part of the 16:0 is also located in the sn-2 position (11). In the bovine sample, all of the 4:0, 93% of the 6:0, and 63% of the 8:0 are esterified to the sn-3 position. With additional analyses, such as determination of the relative molecular weights (carbon numbers) of the TG by GLC, several individual TG in the milks have been tentatively identified. These are: bovine milk (34): rac-14: 0-18:0-18:1, rae- 14:0-18:1-18:0 or rae-18: 0-14:0-18:1, sn- 1,2 (18:0-18:1, 16:0, 4:0, 6:0, or 8:0; and human milk (38): sn-18:0-16:0-14:
0, sn-18:0-l6:O-l6:0, sn-18:1-l6:O-l8:1, sn-18: 1-16:0-18:2, and sn-18:1-18:l-18:2. Obviously, the structures are distinctive. Most of the fatty acids in human milk lipids will eventually, enter the enterocytes via the micellar pathway. The fatty acids up through C12; human, 10.2 mol/lOO mol and bovine, 25.9 mol/loo mol (Table 7), will pass into the portal vein, some through the stomach wall, and the remainder into the enterocyte independent of the micellar pathway. These shorter acids are then carried to the liver where they are quickly oxidized, providing energy. The metabolism of bovine milk TG is unique since no other dietary lipid consumed in any quantity is divided to the extent above, Le., about 25 moll 100 mol of the fatty acids bypassing the micellar pathway of intestinal absorption. This aspect of lipid metabolism has not been investigated. In both milks, the emulsified globules with large surface area are an ideal substrate for lipases (9, 59). Most fluid bovine milk is homogenized, which increases the number of globules and the surface area, providing more space for enzyme activity. In fact, the high coefficients of absorption of lipids in both milks are due in part to the vast surface area of emulsified globules (Table 5). The lipids in infant formulas are also homogenized. The effects of this treatment should be considered when comparative absorptions are studied, but this has not been done. The other factors controlling absorbability are compartmentation in the milks, condition of the intestinal surface, and activities of the lipases and related compounds. We will not discuss the intestinal surface. The TG in both milks are hydrolyzed by gastric and lingual (20) and pancreatic lipases (67). In human milk, the bile salt-stimulated lipase (BSSL) is very active in the breastfed infant's intestine (52). The globule and membrane then have profound effects. In addition to the items mentioned, they carry cholesterol, fat-soluble vitamins, flavor compounds, and other materials soluble in fat. In summary, the globule and membrane are vehicles and compartments, carrying and providing milk components as needed in timed sequences. PHOSPHOLIPIDS
The composition of the milk phospholipids are listed in Table 8 (6, 34, 36, 45. 46, 55). As Jownal of Dairy Science Vol. 73,
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mentioned earlier, these originated from the membranes of the mammary cells and in fresh quiescent milk will be found mostly in the MFGM. Depending upon the severity of the treatment, more or less material will relocate into the skim milk. In addition to their functions as emulsifiers, and MFGM stabilizers, the phospholipids are a source of long-chain PUFA. They bind cations, and possibly interact with digestive enzymes. Because of their location and PUFA content, they are foci of autoxidation. The gangliosides inhibit enterotoxins from Vibrio cholerae and Escherichia coli even though they are present in very small quantities (45). These lipids are one of the many host defense mechanisms in human milk and explain, in part, why in some breastfed infants, the incidence and severity of diarrhea is less than in their nonbreast fed peers. The quantity of active ganglioside, GMt, is much greater in human (12 Ilg/L) than in bovine (1 Ilg/L) milk. The other microphospholipids may also have. as yet, undiscovered nonnutritive functions. STEROLS
The major sterol in both milks is cholesterol in amounts ranging from 10 to 20 mg/dl (34, 36) with the quantities related to the fat content. Trace amounts of other sterols are present, e.g., lanosterol in bovine milk and desmosterol
TABLE 8. Compositions of phospholipids in bovine and human milk lipids. Phospholipid Phosphatidy lcholine Phosphatidy lethanolamine Phosphatidy lserine Phosphatidylinosiliol Sphingomyelin Lysophosphalidy lcholine Lysophosphatidylethanolamine Plasmalogens Diphosphalidylglycerol Ceramides Cerebrosides Gangliosides. mg/L
- (g/IOO g phospholipid) 33.8 27.5 36.3 19.9 3.9 8.4 5.3 5.2 20.8 38.9 Trace Trace Trace
Trace
3.0 Trace Trace Trace 11 3
Trace Trace 11 3
tFrom (34, 36, 46, 55). 2From (6). 3From (45). Journal of Dairy Science Vol. 73.
No.2, 1990
(15) and some phytosterols (23) in human milk. Whole bovine milk is obviously not a high cholesterol food, containing at the most about 200 mg/L. It is unfortunate that the importance of bovine milk as a source of B vitamins, vitamin A, calcium, and high quality protein has been overshadowed by "cholesterolphobia". Recent work by McNamara et a1. (50) indicated that dietary cholesterol had little influence on the plasma cholesterol of their subjects over a 6-wk period. In 69% of the subjects, increased cholesterol intake was accompanied by a decrease in cholesterol fractional absorption or endogenous cholesterol synthesis. The 50 male subjects were asymptomatic for cardiovascular disease and free of secondary causes of hyperlipidemia. Diets rich in saturated fatty acids, (polyunsaturated (P); saturated (S) ratio of .3) as compared with one high in polyunsaturated fatty acids (P:S ratio of 1.5), resulted in higher plasma cholesterol levels. It appears that the impact of diet on plasma cholesterol is individualized; saturated fatty acids are more important than cholesterol in most of the subjects studied. We will discuss the effects of fatty acids on plasma cholesterol levels later. However, in only 30% of the small, healthy group of male subjects studied, endogenous cholesterol synthesis was not inhibited by dietary cholesterol. The type of dietary fat had a large and more consistent effect on plasma cholesterol than dietary cholesterol. Therefore, the fatty acid composition of dietary fats must be considered. Based on the small number of healthy subjects in this study, it would appear that only about 30% of the population would need to severely limit dietary cholesterol. However, the amount and kind of dietary fat still needs attention [See (44, 62) for reviews]. FATTY ACIDS
The fatty acids in both milks have been analyzed extensively with GLC. Analysis of bovine milk fatty acids is difficult because of problems concerning loss and separation of the short-chain fatty acid esters. These have been overcome by the use of temperature programming of the instrument (31, 40) and conversion of the fatty acids to butyl instead of methyl esters (13, 31. 40, 53). Excellent resolution of many fatty acids, including trans isomers, can
231
MILK LIPIDS
TABLE 9. Fatty acid composition of bovine and human milks.
Fany acid'
Bovine2
- - - (g/I00 g fat) - - 3.3 4:0 Trace 6:0 I~ Trace 8:0 1.3 10:0 3.0 1.3 12:0 3.\ 3.1 14.2 5.1 \4:0 \5:0 1.3 Trace to .4 16:0 42.7 20.2 16:1 3.7 5.7 6.0 18:0 5.7 46.4 18:1 16.7 13.0 18:2 1.6 1.4 18:3 1.8 36.1 Tow saturates 76.2 28.8 Hypercholesterolemic4 61.3 1Designated by carbon chain length and number of double bonds. 2From (36). 3From (34).
~otaI of hypercholesterolemic fatty acids (12:0 + 14:0 + 15:0 + 16:0).
be obtained with regular capillary or wide bore capillary columns. These can and should be used routinely. The compositions of the major fatty acids, determined with packed columns, are given in Table 9 (34. 36). Note that as compared with bovine milk, human milk fatty acids have little 4:0 to 8:0, less saturated fatty acids, and more 18:1 and 18:2. Human milk
also contains more long-chain PUFA, which are not listed here. It has been customary to report fatty acid compositions in weight percent, (g of fatty acid/UK) g fat) or normalized data. This is satisfactory when only the fatty acid composition is needed, but if any type of intervention is done that produces a real change in one or more acids, the relative weight percents of the others must change artificially to total 100%. These changes could be misleading because they are not real. Intervention studies should be reported as gravimetric data (mg of fatty acid/ 100 ml milk) (48). Gravimetric data can be determined by use of an internal standard during GLe analysis or by calculation from the fat content. Gravimetric data are a must for the nutritionist. See Table 10 for a comparison of the methods (22). On d 1 of lactation, the sample contained 739.9 mg of 18:1/100 ml or 38.7% of the total fat. At d 30, the amount was 1267.7 mg, but the weight percent had decreased to 35. It is very important to use gravimetric data for human milk, because individual samples are almost always analyzed, and the fatty acid composition responds to dietary changes. It is also important to know the volume of milk involved. Unless an intervention study is being planned which might affect the fatty acid profile and where gravimetric data should be used, the method of reporting is not a problem with bovine milk fatty acids. This is because most market milk has been extensively pooled and will have about the same fat content.
TABLE 10. The fatty acid com~ition of human milk as mg of fatty acids per 100 mI milk (gravimetric)1 and g of fany acid per 100 g fat (normalized).2 Day of lactation
~~ 12:0 14:0 16:0 18:0 18:1 18:2 22:603
8
I
13.4 1 65.8 455.8 179.8 739.9 184.5 4.2
1.1 2
4.3 26.2 9.3 38.7 9.7 .2
93.8 1 163.3 769.3 294.3 1244.0 365.1 8.7
3.~ 5.9 24.8 8.6 36.4 10.8 .3
30 140.5' 210.7 758.9 305.1 1267.7 423.4 5.7
5.5 2 7.2 23.1 8.4 35.0 11.8 .2
lMilligrams of fatty acid per 100 ml milk. Adaple
