Changes in Milk Fat Phospholipids During Lactation JOEL BITMAN and D. L. WOOD Milk Secretion and Mastitis Laboratory Livestock and Poultry SCiences Institute US Department of Agriculture, ARS Beltsville. MD 20705 ABSTRACT
Changes in lipid composition were studied in milk obtained on postpartum d 3 (colostrum), 7, 42, and 180 from 12 Holstein cows. Triglycerides. 96 to 97% of total lipids. were relatively constant during lactation. Phospholipids and cholesterol declined with advancing lactation. Concentrations of the fatty acids synthesized within the mammary gland. CIO:0 to CI6:(). increased about 50% from 7 10 42 d of lactation. During this period. compensatory decreases were observed in C I 8:1. The phospholipids were separated into five major classes: sphingomyelin. phosphatidyl choline. serine, inositol, and ethanolamine for fatty acid analysis. The changes that occurred in milk total fatty acids were reflected in phosphatidyl phospholipid fatty acid composition: an increase in medium-chain fatty acids and a decrease in polyunsaturated fatty acids of 18, 20. and 22 carbon atom chain length as lactation progressed. These changes are consistent with the theory that milk phospholipids are synthesized de novo entirely in the mammary gland. (Key words: phospholipids, cows, fatty acids) INTRODUCTION
The phospholipids (PL) are quantitatively minor constituents of milk fat (1 % of total lipids), but are important structural components of the milk fat globule membrane surrounding the core triglycerides (TO). The PL are present in milk in five major subclasses: sphingomyelin (SM), phosphatidyl choline (PC), phosphatidyl serine (PS), phosphatidyl inositol (PI), and
Received September 20. 1989. Accepted December 11. 1989. \990 J Dairy Sci 73:\208--\2\6
phosphatidyl ethanolamine (PE), each containing 25 to 30 fatty acids (FA). The relatively small amount of PL in milk in the presence of extremely large amounts of lipids present as TO (98% of total lipids) presents a major analytical challenge when milk supplies are limited. Because of the abundant production and ready availability of cows' milk. the detailed composition of individual PL has been determined, primarily from isolation by column chromatography of the subclasses from batches of spray-dried buttermilk powders (13. 14. 15). However, to the best of our knowledge, changes that occur in PL FA during lactation in the cow have not been characterized. We have recently developed analytical methods for the accurate determination of PL which are applicable to small volumes of human breast milk (2. 3). These methodologies involve isolation of the PL classes by quantitative TLC followed by capillary column OLe. Because milk PL appear to be synthesized de novo entirely in mammary tissue (7, 17), examination of milk PL FA and total milk FA composition could provide information on relative rates of synthesis of important milk constituents (milk fat globule membranes and core lipids). Specific objectives of the present study were 1) to investigate PL FA compositional changes occurring as lactation progressed from d 3 to 180. and 2) to relate these changes to the major changes that occur in the FA of milk fat during lactation. MATERIALS AND METHODS
Twelve Holstein cows were assigned to this experiment. They were housed in tie stalls and machine milked twice daily. Cows were offered a total mixed ration at rates based upon milk production. The total mixed ration was a 60:40 forage:concentrate mix containing ground shelled com, alfalfa silage, com silage, and concentrate. A 250- to 3OQ-ml milk sample was
1208
1209
MILK FAT PHOSPHOLIPIDS DURING LACTATION
obtained from the total a.m. milking of each cow on postpartum d 3 (colostrum), d 7 (transitional milk), d 42 (mature milk), and d 180 (midlactation milk). Within 30 min of milking, samples were stored at -20'C until analyzed. Frozen samples were heated rapidly to 80'C and held for 1.5 min to inactivate lipases (19). Lipid wa" extracted from the milk sample with chlorofonn-methanol (2: 1) by the Folch procedure (10). Neutral lipid (NL) and PL classes were determined by quantitative densitometric TLC (2, 3). The NL were separated by preparative TLC (3) into the following classes: TO, 1,2-diglycerides (1,2-00), FFA, monoglycerides (MO), cholesterol (C), and cholesteryl esters (CE). Phospholipids were separated by preparative TLC (3) into their subclasses: SM, PC. PS, PI, and PE. Total FA were detennined by OLC of fany acid methyl esters (2). Fatty acids results are expressed as weight percent (wt/wt) of total FA. Total lipid was detennined gravimetrically. Cholesterol and CE were detennined by quantitative densitometry in situ following separation by two-stage TLC (4, 21). All data were statistically analyzed by Fisher's protected least significant differences (5).
tribution of lipids among the individual classes during lactation have not been reported before and are shown in Table 1. Triglycerides, the major component of the milk fat globule core, constituted 96 to 97% of total lipids, and were relatively constant during lactation. Phospholipids and C were present in concentrations of 1.0 and .5% of total milk fat lipids. Cholesteryl esters composed only about 10% of the C fraction (about .05% of total lipid). Cholesterol and PL declined with advancing lactation. Patton and Jensen (17) indicated that approximately 60% of the PL and 85% of the C is associated with milk fat globules. A decline in these two membrane constituents was also observed in human milk as lactation progressed (2). Kinsella and Houghton (12) found that total PL secretion decreased during lactation in cows fed either nonnal or low fiber diets. Changes Occurring In Fatty Acids During Lactation
The OLC analysis of the FA methyl esters indicated that over 95% of the lipid was present in 12 major FA from C4:0 to C 18:3 (C4 :0' C6:0, C8:O, ClO:O' C12:o, C14:0, C 16:0' CI6:), C18:0' C I8:), C18:2, CI8:3; Table 2). Two major changes occurred as lactation progressed: 1) the medium-chain FA, ClO:0 to CI4:0' increased markedly from colostrum to mature milk (+50%), and 2) consistent compensatory decreases were noted in concentrations of stearic (C I8:0), oleic (C I8:I), and linoleic (CI8:2) acids in mature milk (Table 2). Oleic acid decreased
RESULTS AND DISCUSSION
Changes Occurring In Lipid Classes During lactation
Total fat content ranged from 3.1 to 4.0% and exhibited a trend toward lower values as lactation progressed (Table I). Changes in dis-
TABLE 1. Lipid class composition of cows' milk during lactation. Percentage at lactation day Lipid class t
Pooled SE
7
42
180
1.11· .46 b 95.SO b
1.2-00 FFA MG CE
1.06· .4l b 97.11" 1.16c .19 .06 .03
2.25 a
.56b .30" 97.l7 ab I.nb
.09
.53· 97.35" 1.0l c .26 .06 .05
.28 .08 .02
.18 .03 .04
.04
Fat. g/d1 Number of cows
3.45 12
3.97 12
3.25 12
3.13 12
.24
3
.nb
PL C
TG
.03 .15 .12 .01
.01
•.b,CMeans within a row with different superscripts differ (P were the predominant FA and made up about two-thirds of total FA. The medium-chain FA, C12:o, C I4 :0, and C 16:0 (half of which is synthesized in the mammary gland), all increased as lactation progressed. The major FA not synthesized in the mammary gland, C18:1 and CAI8:2, decreased as lactation progressed, but the concentration of C18:0 was unchanged. Decreases were also observed in arachidonic acid, C20:4' and in C22:5 and C22:6 (Table 4). We found approximately equal amounts of saturated and unsaturated FA in PC, which is in general agreement with the data of
TABLE 3. Phospholipid composition of cows' milk during lactation. Concentration at lactation day. mg/dl Class l SM PC PS PI PE Total SM PC PS PI PE
3 5.8b 5.8 cd 1.6b .8 b 6.4b 20.4 28.7 b 28.0b 8.l a 4.l d 3l.aa
7
42
11.9a 8.9 a 3.0a 1.6" 10.Oa 35.4
7.1 b 6.7 bc 2.1 b 1.3a 7.~ 25.1
34.1 a 25.1 b 8.4 a 4.6 cd 27.g a
Pooled SE
18O 3.9c 4.5 d .3 c 1.5 a 2.6c
.5 .6 .2 .1 .8 2.0
12.8
Percentage of phospholipids 28.7 b 31.4 ab 35.l a 26.4b 1.9b 8.5a 11.8 a 5.2bc 3 1.1 a 19.8 b
1.1 1.1 .4 .3 1.6
a,b.CMeans within a row with different superscripts differ (P and CI8:2' Significant changes were observed as lactation progressed, the medium-chain FA in-
TABLE 4. Fatty acid composition Fan~
acid
3
12:0 14:0 14:1 15:0 16:0br 16:0 16:1 17:0 18:0br 18:0 18:1 18:2 20:0 18:3 21:0 20:3 20:4 20:5 22:4
.lb 2.7 b .2c .8c Abe 28.3 b 3.l b I.I b .8 I 1.1 a 33.7 a 1O.4be .5 1.2 .4
22:5006
22:5003 22:6 PUFA 3 Saturated
1.1
2.1" A
.5 5 .5 ab .1 a 16.8 a 46.2 b
(WI %)
of phosphatidyl choline of bovine milk. Percentage at lactation day 7 42 180 b a .2 .4a 3 4.l b 6.7 a 7.l a .3c .6 b .7a .'1' 1.5 b 2.0.3c .6a .5ab 28.3b 32.2a 31.7a 3.7a 3A b 3Ab .'1' 1.0be 1.2a .7 7 .6 8.3 b 7.5 b 11.2a b 30.l 34.3" 30.l b IU a 7.4 d 8.'f'd A .4 A 1.2 104 1.1 .4 .3 .0 1.1 1.0 1.0 2.0a 1.2b .7c .6 .5 .1 .4 .2 .3 .3 A .2 .6a .3 be .2c .Ob .Ob .Ob I J.(f 17.3 a 14.l b 44.5 b 51.6a 54.7a
a,b.cMeans within a row with different superscripts differ (P
Changes in lipid composition were studied in milk obtained on postpartum d 3 (colostrum), 7, 42, and 180 from 12 Holstein cows. Triglycerides. 96 to 97% of total lipids. were relatively constant during lactation. Phospholipids and cholesterol declined with advancing lactation. Concentrations of the fatty acids synthesized within the mammary gland. CIO:0 to CI6:(). increased about 50% from 7 10 42 d of lactation. During this period. compensatory decreases were observed in C I 8:1. The phospholipids were separated into five major classes: sphingomyelin. phosphatidyl choline. serine, inositol, and ethanolamine for fatty acid analysis. The changes that occurred in milk total fatty acids were reflected in phosphatidyl phospholipid fatty acid composition: an increase in medium-chain fatty acids and a decrease in polyunsaturated fatty acids of 18, 20. and 22 carbon atom chain length as lactation progressed. These changes are consistent with the theory that milk phospholipids are synthesized de novo entirely in the mammary gland. (Key words: phospholipids, cows, fatty acids) INTRODUCTION
The phospholipids (PL) are quantitatively minor constituents of milk fat (1 % of total lipids), but are important structural components of the milk fat globule membrane surrounding the core triglycerides (TO). The PL are present in milk in five major subclasses: sphingomyelin (SM), phosphatidyl choline (PC), phosphatidyl serine (PS), phosphatidyl inositol (PI), and
Received September 20. 1989. Accepted December 11. 1989. \990 J Dairy Sci 73:\208--\2\6
phosphatidyl ethanolamine (PE), each containing 25 to 30 fatty acids (FA). The relatively small amount of PL in milk in the presence of extremely large amounts of lipids present as TO (98% of total lipids) presents a major analytical challenge when milk supplies are limited. Because of the abundant production and ready availability of cows' milk. the detailed composition of individual PL has been determined, primarily from isolation by column chromatography of the subclasses from batches of spray-dried buttermilk powders (13. 14. 15). However, to the best of our knowledge, changes that occur in PL FA during lactation in the cow have not been characterized. We have recently developed analytical methods for the accurate determination of PL which are applicable to small volumes of human breast milk (2. 3). These methodologies involve isolation of the PL classes by quantitative TLC followed by capillary column OLe. Because milk PL appear to be synthesized de novo entirely in mammary tissue (7, 17), examination of milk PL FA and total milk FA composition could provide information on relative rates of synthesis of important milk constituents (milk fat globule membranes and core lipids). Specific objectives of the present study were 1) to investigate PL FA compositional changes occurring as lactation progressed from d 3 to 180. and 2) to relate these changes to the major changes that occur in the FA of milk fat during lactation. MATERIALS AND METHODS
Twelve Holstein cows were assigned to this experiment. They were housed in tie stalls and machine milked twice daily. Cows were offered a total mixed ration at rates based upon milk production. The total mixed ration was a 60:40 forage:concentrate mix containing ground shelled com, alfalfa silage, com silage, and concentrate. A 250- to 3OQ-ml milk sample was
1208
1209
MILK FAT PHOSPHOLIPIDS DURING LACTATION
obtained from the total a.m. milking of each cow on postpartum d 3 (colostrum), d 7 (transitional milk), d 42 (mature milk), and d 180 (midlactation milk). Within 30 min of milking, samples were stored at -20'C until analyzed. Frozen samples were heated rapidly to 80'C and held for 1.5 min to inactivate lipases (19). Lipid wa" extracted from the milk sample with chlorofonn-methanol (2: 1) by the Folch procedure (10). Neutral lipid (NL) and PL classes were determined by quantitative densitometric TLC (2, 3). The NL were separated by preparative TLC (3) into the following classes: TO, 1,2-diglycerides (1,2-00), FFA, monoglycerides (MO), cholesterol (C), and cholesteryl esters (CE). Phospholipids were separated by preparative TLC (3) into their subclasses: SM, PC. PS, PI, and PE. Total FA were detennined by OLC of fany acid methyl esters (2). Fatty acids results are expressed as weight percent (wt/wt) of total FA. Total lipid was detennined gravimetrically. Cholesterol and CE were detennined by quantitative densitometry in situ following separation by two-stage TLC (4, 21). All data were statistically analyzed by Fisher's protected least significant differences (5).
tribution of lipids among the individual classes during lactation have not been reported before and are shown in Table 1. Triglycerides, the major component of the milk fat globule core, constituted 96 to 97% of total lipids, and were relatively constant during lactation. Phospholipids and C were present in concentrations of 1.0 and .5% of total milk fat lipids. Cholesteryl esters composed only about 10% of the C fraction (about .05% of total lipid). Cholesterol and PL declined with advancing lactation. Patton and Jensen (17) indicated that approximately 60% of the PL and 85% of the C is associated with milk fat globules. A decline in these two membrane constituents was also observed in human milk as lactation progressed (2). Kinsella and Houghton (12) found that total PL secretion decreased during lactation in cows fed either nonnal or low fiber diets. Changes Occurring In Fatty Acids During Lactation
The OLC analysis of the FA methyl esters indicated that over 95% of the lipid was present in 12 major FA from C4:0 to C 18:3 (C4 :0' C6:0, C8:O, ClO:O' C12:o, C14:0, C 16:0' CI6:), C18:0' C I8:), C18:2, CI8:3; Table 2). Two major changes occurred as lactation progressed: 1) the medium-chain FA, ClO:0 to CI4:0' increased markedly from colostrum to mature milk (+50%), and 2) consistent compensatory decreases were noted in concentrations of stearic (C I8:0), oleic (C I8:I), and linoleic (CI8:2) acids in mature milk (Table 2). Oleic acid decreased
RESULTS AND DISCUSSION
Changes Occurring In Lipid Classes During lactation
Total fat content ranged from 3.1 to 4.0% and exhibited a trend toward lower values as lactation progressed (Table I). Changes in dis-
TABLE 1. Lipid class composition of cows' milk during lactation. Percentage at lactation day Lipid class t
Pooled SE
7
42
180
1.11· .46 b 95.SO b
1.2-00 FFA MG CE
1.06· .4l b 97.11" 1.16c .19 .06 .03
2.25 a
.56b .30" 97.l7 ab I.nb
.09
.53· 97.35" 1.0l c .26 .06 .05
.28 .08 .02
.18 .03 .04
.04
Fat. g/d1 Number of cows
3.45 12
3.97 12
3.25 12
3.13 12
.24
3
.nb
PL C
TG
.03 .15 .12 .01
.01
•.b,CMeans within a row with different superscripts differ (P were the predominant FA and made up about two-thirds of total FA. The medium-chain FA, C12:o, C I4 :0, and C 16:0 (half of which is synthesized in the mammary gland), all increased as lactation progressed. The major FA not synthesized in the mammary gland, C18:1 and CAI8:2, decreased as lactation progressed, but the concentration of C18:0 was unchanged. Decreases were also observed in arachidonic acid, C20:4' and in C22:5 and C22:6 (Table 4). We found approximately equal amounts of saturated and unsaturated FA in PC, which is in general agreement with the data of
TABLE 3. Phospholipid composition of cows' milk during lactation. Concentration at lactation day. mg/dl Class l SM PC PS PI PE Total SM PC PS PI PE
3 5.8b 5.8 cd 1.6b .8 b 6.4b 20.4 28.7 b 28.0b 8.l a 4.l d 3l.aa
7
42
11.9a 8.9 a 3.0a 1.6" 10.Oa 35.4
7.1 b 6.7 bc 2.1 b 1.3a 7.~ 25.1
34.1 a 25.1 b 8.4 a 4.6 cd 27.g a
Pooled SE
18O 3.9c 4.5 d .3 c 1.5 a 2.6c
.5 .6 .2 .1 .8 2.0
12.8
Percentage of phospholipids 28.7 b 31.4 ab 35.l a 26.4b 1.9b 8.5a 11.8 a 5.2bc 3 1.1 a 19.8 b
1.1 1.1 .4 .3 1.6
a,b.CMeans within a row with different superscripts differ (P and CI8:2' Significant changes were observed as lactation progressed, the medium-chain FA in-
TABLE 4. Fatty acid composition Fan~
acid
3
12:0 14:0 14:1 15:0 16:0br 16:0 16:1 17:0 18:0br 18:0 18:1 18:2 20:0 18:3 21:0 20:3 20:4 20:5 22:4
.lb 2.7 b .2c .8c Abe 28.3 b 3.l b I.I b .8 I 1.1 a 33.7 a 1O.4be .5 1.2 .4
22:5006
22:5003 22:6 PUFA 3 Saturated
1.1
2.1" A
.5 5 .5 ab .1 a 16.8 a 46.2 b
(WI %)
of phosphatidyl choline of bovine milk. Percentage at lactation day 7 42 180 b a .2 .4a 3 4.l b 6.7 a 7.l a .3c .6 b .7a .'1' 1.5 b 2.0.3c .6a .5ab 28.3b 32.2a 31.7a 3.7a 3A b 3Ab .'1' 1.0be 1.2a .7 7 .6 8.3 b 7.5 b 11.2a b 30.l 34.3" 30.l b IU a 7.4 d 8.'f'd A .4 A 1.2 104 1.1 .4 .3 .0 1.1 1.0 1.0 2.0a 1.2b .7c .6 .5 .1 .4 .2 .3 .3 A .2 .6a .3 be .2c .Ob .Ob .Ob I J.(f 17.3 a 14.l b 44.5 b 51.6a 54.7a
a,b.cMeans within a row with different superscripts differ (P
