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Positional Distribution of Fatty Acids in Triglycerides from Prepartum Mammary Gland Secretion and Early Postpartum Milk
Positional Distribution of Fatty Acids in Triglycerides from Prepartum Mammary Gland Secretion and Early Postpartum Milk PETER W. P A R O D I Fats Research Laboratory The Butter Marketing Board Hamilton Central, 4007, Queensland Australia
ABSTRACT
Parodi (19) did not find large variation in stereospecific distribution of fatty acids in butter throughout the dairying season. To obtain milk fat samples that exhibited larger variation in fatty acid content, Parodi (19) placed a cow on a restricted feeding regimen. Restricted feeding decreased content of short- and medium-chain fatty acids and concurrently increased 18:1 acids of the triglycerides. This was not associated with a random increase or decrease of acids at the three stereospecific positions. A specific mechanism operated, namely, with changing triglyceride fatty acid content (6:0, 8:0, 10:0, 12:0, 14:0, and 16:0 decreasing and 18:1 increasing)the proportions of 6:0, 8:0, 10:0, 12:0, 14:0, and 16:0 acids at the sn-3-position decreased whereas the proportions of 18:0 and 18:1 acids increased. Changes of proportions of individual acids at the sn-3-position were compensated for by changes in the opposite direction at the sn-1and sn-2-positions. Triglyceride structure of prepartum mamma W gland secretion and early postpartum milk is examined for further knowledge of variation in positional distribution of fatty acids with changing triglyceride composition.
Triglycerides from mammary gland secretion collected at 24, 17, 10, 7, and 3.5 days prepartum and from milk collected at 0 (2 h), 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 days postpartum were subjected to fatty acid and stereospecific analysis. Prepartum mammary gland secretion differed from normal milk in that it contained approximately twice the amount of 16:0 and larger than normal amounts of 14:0. This was compensated for by less amounts of other mediumchain, short-chain, 18:0, and 18:1 acids. Positional distribution of fatty acids in milk from 1 day postpartum onward generally followed the specific pattern normally encountered for milk triglycerides. With exception of the first sample at 24 days prepartum, mammary gland secretion had medium-chain acids at 8:0, 10:0, 12:0, and sometimes 14:0 preferentially esterified at the sn-3position rather than the sn-2-position. Relationships were linear between content of fatty acids at the three stereospecific positions and content of the same acid in triglycerides. These significant relationships are represented by regression equations. With varying triglyceride fatty acid content, fatty acid content at the three positions did not change at the same rate.
MATERIALS AND METHODS Samples
INTRODUCTION
Although fatty acid composition of butter varies seasonally (6, 17), range of variation for individual fatty acids is not large. This may explain why Taylor and Hawke (23) and Received December 18, 1981.
1983 J Dairy Sci. 66:912--919
912
Secretion from mammary gland at 24, 17, 10, 7, and 3.5 days prepartum was obtained from a multiparous pregnant Friesian cow determined to be free from clinical or subclinical mastitis by the Wisconsin Mastitis Test (24). The total secretion was collected from all quarters, except at 3.5 days prepartum when only an aliquot was collected from each quarter. Lipid was extracted from the secretion by the method of Folch et al. (5). Milk samples were collected at 0 (2 h), 1, 2,
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TECHNICAL NOTE 3, 4, 5, 6; 7, 14, 21, and 28 days postpartum. Lipid was obtained by the Roese-Gottlieb method (1). Lipid was stored under nitrogen at ~20°C until required for analysis. Isolation of Triglycerides
Triglycerides were obtained from crude lipid by column chromatography with 7% hydrated Florisil (8). Stereospecific Analysis
The sn-l,2 (2,3)-diglyceride method of Brockerhoff (2), adapted for milligram quantities by Christie and Moore (3), was used with modification. This method together with the pancreatic lipase deacylation procedure used to obtain mono- and diglycerides was reported by Parodi (19). A sample of interesterified milk fat was used to obtain optimum conditions for pancreatic lipase deacylation. Fatty Acid Analysis
Glycerides and partial glycerides were transesterified to methyl esters by the method of Shehata et al. (21). Phospholipids generated during stereospecific analysis were transesterifled by the addition of 5/11 of 2.0 N methanolic sodium methoxide and 50 /al of hexane followed by shaking for 5 rain. Fatty acid methyl esters were analyzed by gas liquid chromatography (GLC) as outlined by Parodi (17, 18). RESULTS
Until 3.5 days prepartum the cow only produced about 50 ml of secretion per sampling period. Although not measured quantitatively, the secretion contained far less lipid than normal milk. Lipids from the secretion and milk were fractionated by column chromatography on Florisil to obtain the triglycerides, which were subjected to fatty acid and stereospecific analysis. Trigtyceride fatty acid composition is in Table 1. Triglycerides from prepartum mammary gland secretion differed from those of normal milk in that they contained approximately twice the 16:0 acid and larger than normal amounts of 14:0. This was compensated for by less 4:0, 6:0, 8:0, 10:0, 18:0, and 18:1 fatty acids. The positional distribution of fatty acids in triglycerides from prepartum mammary gland
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23.7 20.3 35.7
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22.8 6.6 11.9
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secretion and postpartum milk is in Table 2. The distribution of individual fatty acids in milk from 1 day postpartum onward generaIly followed the specific pattern normally encountered for milk fat. The 4:0 and 6:0 fatty acids were esterified almost exclusively at the sn-3-position. The 8:0, 10:0, 12:0, and 14:0 acids were located preferentially at the sn-2 position; 16:0 was almost equally distributed between the sn-1- and sn-2-positions, and the 18:0 acid was esterified selectively at the sn-1position. The 18:1 acid usually is distributed roughly equally between the sn-1 and sn-3positions (19), but with the cow under study there was relatively less 18:1 at the sn-1position. With exception of the first sample at 24 days prepartum, mammary gland secretion had medium-chain acids 8:0, 10:0, 12:0, and sometimes 14:0 preferentially esterified at the sn-3position rather than the sn-2-position. There was also a tendency for 18:0 to be selectively esterified at the sn-2- or sn-3-positions rather than the sn-l-position. These abnormalities disappeared by the 1st day postpartum. Variation in content of individual fatty acids (10:0, 12:0, 14:0, 16:0, 18:0, and 18:1) at each of the three positions was compared with variation in total content of corresponding acids in triglycerides by regression equations. Equations were in the form y = m x + c; where y = content of fatty acid at position sn-1 or sn-2 or sn-3-; x = content of the same fatty acid in triglycerides; m and c arc constants. When c was not significantly different from zero (P<.05), the equation was in the form y=mx. Contents for 10:0 and 12:0 at the sn-2and sn-3-positions of prepartum mammary gland secretion were not included because of their abnormal distribution referred to earlier. Linear regression equations and correlation coefficients for the comparisons are in Table 3. Over the range examined, linear relationships, generally with highly significant correlation coefficients, were found between content of a fatty acid at all three positions and content of the same acid in the intact triglycerides. As the content of an acid increased in the triglycerides, there were increases in the content of that acid at the three positions. With varying triglyceride fatty acid content, the fatty acid content at Journal of Dairy Science Vol. 66, No. 4, 1983
the three positions did not change in an uniform manner. From Table 3 for the 18:1, 16:0, and 14:0 acids the change in content at the sn-3-position was more pronounced than at the sn-1- and sn-2-positions. The change in content of 18:0 was most marked at the sn-1position, and changes in 12:0 and 10:0 were more pronounced at the sn-2-position. DISCUSSION
The small quantity of mammary gland secretion produced until 3.5 days prepartum and its low content of lipid are in line with findings of Wheelock et al. (25) and Smith et al. (22), who also noted differences between animals in the amount of secretion produced. The first sample, at 24 days prepartum, had a different fatty acid composition from those at later prepartum periods. Its composition would be consistent with that of a sample of prepartum secretion diluted by milk from a previous lactation. Rowland et al. (20) found little carryover of secretion from the end of one lactation to the beginning of the next. However, little carry-over milk would have been needed to give the observed fatty acid composition. High concentrations of 16:0 and 14:0, significant amounts of 12:0 and 10:0, and low content of 18:0 in prepartum mammary gland secretion are in line with findings of Mellenberger et al. (18) and Kinsella (9). These workers measured compositions of fatty acids synthesized from [14Clacetate by mammary gland slices and homogenates obtained by biopsy from cows at various times before parturition. The other significant feature of the fatty acid composition of mammary gland secretion is the monoene fatty acids. The secretion contained higher than normal concentrations of 14:1 and 16:1 acids (confirmed by hydrogenation) and less than normal amount of 18:1. The question arises, did these acids originate from plasma triglycerides, from desaturation in the mammary gland, or from both sources? The presence of odd carbon-chain and branchedchain acids indicated plasma triglycerides were used as a source of fatty acids for milk fat synthesis. The higher contents of 14:1 and 16:1 acids corresponded to the high concentrations of 14:0 and 16:0, whereas the low content of 18:1 corresponded to the low content of 18:0. It is possible that the monoene acids resulted
TECHNICAL NOTE f r o m desaturase activity in t h e m a m m a r y gland. Kinsella and Heald (12) r e p o r t e d stearyl desaturase activity in b o v i n e m a m m a r y tissue o b t a i n e d 2 days b e f o r e p a r t u r i t i o n b u t n o t b y m a m m a r y tissue o b t a i n e d 1 to 2 wk b e f o r e p a r t u r i t i o n . T h e small a m o u n t of in vivo triglyceride s y n t h e s i s in t h e c u r r e n t s t u d y at 3.5 t o 2 4 days p r e p a r t u m w o u l d require little d e s a t u r a s e activity. This m a y n o t have b e e n d e t e c t e d in the s t u d y of Kinsella and Heald (12). A f t e r 24 d a y s p r e p a r t u m the 8:0, 10:0, and 1 2 : 0 f a t t y acids were esterified p r e f e r e n t i a l l y at t h e sn-3-position. F o l l o w i n g p a r t u r i t i o n these acids were esterified p r e f e r e n t i a l l y at t h e sn-2p o s i t i o n . T h e c o n t e n t s of these acids at the s n - 2 - p o s i t i o n were far m o r e t h a n c o n t e n t s for 37 samples of b u t t e r (19). W h e n a cow was placed on a restricted feeding regimen (19),
917
milk triglycerides b e f o r e r e s t r i c t e d feeding contained t h e 8 : 0 a n d 1 0 : 0 acids p r e f e r e n t i a l l y esterified at the sn-3-position. A f t e r r e s t r i c t e d feeding the)' were esterified p r e f e r e n t i a l l y at t h e sn-2-position. T h e s e changes do n o t a p p e a r to be related to t h e c o n t e n t of acids in the triglycerides. It is possible t h a t t h e physiological c o n d i t i o n of t h e cow m a y i n f l u e n c e some of t h e factors a f f e c t i n g a c y l a t i o n of f a t t y acids. O b t a i n i n g milk triglyceride samples e x h i b i t ing a larger t h a n n o r m a l range of f a t t y acid c o n t e n t s m a d e it possible to d e m o n s t r a t e t h e linear r e l a t i o n s h i p s b e t w e e n c o n t e n t s o f f a t t y acids at t h e t h r e e p o s i t i o n s and c o n t e n t s o f t h e same acids in the triglycerides. Christie and M o o r e (4) used 45 s a m p l e s o f pig adipose tissue w i t h a wide range of f a t t y acid c o n t e n t s and f o u n d linear r e l a t i o n s h i p s b e t w e e n p o s i t i o n a l d i s t r i b u t i o n of f a t t y acids and the a m o u n t of
TABLE 3. Linear regression analyses and correlation coefficients for the relationship between fatty acid composition at the three positions and the composition of Che same acid in the triglycerides. Fatty acid
Position
Slope
Intercept
r
18:1
sn-1 sn-2 sn- 3
1.31 .71 1.38
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.
.
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1.76 .51 .95
-2.44 . . . .
. .
. .
.977"** 860"* * 901 ** *
sn-1 sn-2 sn-3
1.03 .74 1,19
10.77 13.20 - 22,94
.979* * *
14:0
sn-1 sn-2 sn-3
.66 .52 1.79
1.78 12.49 - 14.00
.967* * * .722** .949* * *
12:0
sn-1 sn-2 sn-3
.73 1.89 .36
.
sn-1 sn-2 sn-3
.38 2.40 .23
. . .
18:0
16:0
10:0
.937** * . 9 4 7 ** *
.893*** .947** * .506
"1".26 -1.21 . . .
. . .
. . .
788** 982* * * 662*
aIn these cases the intercept was not significantly different from zero, and the equation was calculated in the form y = rex. *P<.05. **P<.01. ***P<.001. Journal of Dairy Science Vol. 66, No. 4, 1983
918
PARODI
t h e same acid in the triglycerides. Litchfield (13) has t a k e n d a t a f r o m severat studies of p a n c r e a t i c Iipase d e a c y l a t i o n of seed and m a r i n e oils a n d s h o w n for some acids relationships b e t w e e n c o n t e n t s at t h e sn-2- a n d sn-l,3p o s i t i o n s a n d c o n t e n t s in triglycerides. W h e n c o n t e n t o f an acid c h a n g e d in the triglycerides, t h e c o n t e n t at the t h r e e stereospecific p o s i t i o n s did n o t change at the same rate. T h e f a t t y acid c o m p o s i t i o n of t h e sn-1 a n d s n - 2 - p o s i t i o n s is c o n t r o l l e d b y specificities of the sn-glycerol-3-phosphate and 1-acyl-sng l y c e r o l - 3 - p h o s p h a t e acyltransferases (7, 10, 14). A t t h e s n - 3 - p o s i t i o n t h e f a t t y acid c o m p o s i t i o n does n o t a p p e a r related to t h e specificities of diacylglycerol a c y l t r a n s f e r a s e s b u t r a t h e r is a f u n c t i o n of t h e c o m p o s i t i o n of t h e intracellular acyl-CoA p o o l (15). Kinsella a n d Gross (11) believe t h e r e is s o m e segregation in the m a m m a r y gland of f a t t y acids derived f r o m p l a s m a triglycerides a n d t h o s e s y n t h e s i z e d de n o v o . T h e l a t t e r m a y be utilized largely for a c y l a t i o n at t h e sn-3-position. W h e n a cow was placed o n a restricted feeding regimen (19), t h e r e was a decrease in triglycerides of t h o s e f a t t y acids s y n t h e s i z e d de novo in t h e m a m m a r y gland. T h e s e decreases were greater at t h e s n - 3 - p o s i t i o n t h a n at t h e sn-1 and s n - 2 - p o s i t i o n s a n d r e s u l t e d in triglycerides w i t h o n l y small a m o u n t s of 1 4 : 0 a n d 1 6 : 0 at t h e sn-3-position. Decreased a m o u n t s of de novo s y n t h e s i z e d f a t t y acids at t h e s n - 3 - p o s i t i o n were c o m p e n sated for b y increased c o n t e n t s of 18: 1.
ACKNOWLEDGMENTS I t h a n k Carol Beck f o r t e c h n i c a l assistance and R. E. Times, CSIRO, Division of F o o d R e s e a r c h , Dairy R e s e a r c h L a b o r a t o r y , for statistical analysis. B. E. Wilson o f the Veterin a r y Science F a r m , U n i v e r s i t y of Q u e e n s l a n d , supplied t h e m i l k samples. T h i s w o r k was supp o r t e d in p a r t b y a g r a n t f r o m t h e A u s t r a l i a n Dairy R e s e a r c h C o m m i t t e e .
REFERENCES 1 Association of Official Analytical Chemists. 1970. Official methods of analysis, l lth ed. Assoc. Offic. Anal. Chem., Washington, DC. 2 Brockerhoff, H. 1965. A stereospecific analysis of Journal of Dairy Science Vol. 66, No, 4, 1983
triglycerides. J. Lipid Res. 6:10. 3 Christie, W. W., and J. H. Moore. 1969. A Semimicro method for the stereospecific analysis of triglycerides. Biochim. Biophys. Acta 176:445. 4 Christie, W. W., and J. H. Moore. 1970. The variation of triglyceride structure with fatty acid composition in pig adipose tissue. Lipids 5:921. 5 Folch, J., M. Lees, and G.H.S. Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem.
226:497. 6 Gray, 1. K. 1973. Seasonal variations in the composition and thermal properties of New Zealand milk fat. 1. Fatty acid composition. J. Dairy Res. 40:207. 7 Gross, M. J., and J. E. Kinsella. 1974. Properties of palmityl-CoA: L-~-glycerolphosphate acyltransferase from bovine mammary mierosomes. Lipids 9:905. 8 Kates, M. 1972. Techniques of lipidology: isolation, analysis and identification of lipids. Page 269 in Laboratory techniques in biochemistry and molecular biology. T. S. Work and E. Work, ed. North Holland, Amsterdam. 9 Kinsella, J. E. 1975. Coincident synthesis of fatty acids and secretory triglycerides in bovine mammary tissue. Int. J. Biochem. 6:65. I0 Kinsella, J. E. 1976. Monoacyl-sn-glycerol-3phosphate acyltransferase specificity in bovine mammary microsomes. Lipids 11:680. 11 Kinsella, J. E., and M. Gross. 1973. Palmitic acid and initiation of mammary glyceride synthesis via phosphatidic acid. Biochim. Biophys. Acta 316: 109. 12 Kinsella, J. E., and C, W. Heald. 1972. Nal-14C stearate and Na2-14C acetate metabolism and morphological analysis of the late prepartum bovine mammary tissue. J, Dairy Sci. 55:1085. 13 Litcbfield, C. 1972. Page 233 in Analysis of triglycerides. Academic Press, New York, NY. 14 Marshall, M. O., and J. Knudsen. 1977. The specificity of 1-acyl-sn-glycerol-3-phosphate acyltransferase in microsomal fractions from lactating cow mammary gland towards short, medium, and long chain acyl-CoA esters. Biochim. Biophys. Acta 489:236. 15 Marshall, M. O., and J. Knudsen. 1979. Specificity of diacylglycerol acyltransferase from bovine mammary gland, liver and adipose tissue towards acylCoA esters. Eur. J. Biochem. 94:93. 16 Mellenberger, R. W., D. E. Bauman, and D. R. Nelson. 1973. Metabolic adaptations during lactogenesis. Fatty acid and lactose synthesis in cow mammary tissue. Biochem. J. 136:741. 17 Parodi, P. W. 1970. Fatty acid composition of Australian butter and milk fats. Australian J. Dairy Technol. 25:200. 18 Parodi, P. W. 1972. Observations on the variation in fatty acid composition of milkfat. Australian J. Dairy Technol. 27:90. 19 Parodi, P. W. 1979. Stereospecific distribution of fatty acids in bovine milk fat triglycerides. J. Dairy Res. 46:75. 20 Rowland, S. J., J.H.B. Roy, H. J. Sears, and S. Y.
TECHNICAL NOTE Thompson. 1953. The effect of prepartum milking on the composition of the prepartum and postpartum secretions of the cow. J. Dairy Res. 20:16. 21 Shehata, A. Y., J. M. de Man, and J. C. Alexander. 1970. A simple and rapid method for the preparation of methyl esters of fats in milligram amounts for gas chromatography. Can. Inst. Food Technol. J. 3:85. 22 Smith, A., J. V. Wheelock, and F. H. Dodd. 1967. Changes in the quantity and composition of mammary gland secretion in the dry period between lactations. I1. The complete dry period. J. Dairy
919
Res. 34:13. 23 Taylor, M. W., and J. C. Hawke. 1975. Structural analysis of the triacylglycerols of bovine milkfats. New Zealand J. Dairy Sci. Technol. 10:49. 24 Thompson, D. I., and D. S. Postle. 1964. The Wisconsin Mastiffs T e s t - a n indirect estimation of leucocytes in milk. J. Food Technol. 27:271. 25 Wheelock, J. V., A. Smith, F. H. Dodd, and R.L.J. Lyster. 1967. Changes in the quantity and composition of mammary gland secretion in the dry period between lactations. I. The beginning of the dry period. J. Dairy Res. 34:1.
Journal of Dairy Science Vol. 66, No. 4, 1983
ABSTRACT
Parodi (19) did not find large variation in stereospecific distribution of fatty acids in butter throughout the dairying season. To obtain milk fat samples that exhibited larger variation in fatty acid content, Parodi (19) placed a cow on a restricted feeding regimen. Restricted feeding decreased content of short- and medium-chain fatty acids and concurrently increased 18:1 acids of the triglycerides. This was not associated with a random increase or decrease of acids at the three stereospecific positions. A specific mechanism operated, namely, with changing triglyceride fatty acid content (6:0, 8:0, 10:0, 12:0, 14:0, and 16:0 decreasing and 18:1 increasing)the proportions of 6:0, 8:0, 10:0, 12:0, 14:0, and 16:0 acids at the sn-3-position decreased whereas the proportions of 18:0 and 18:1 acids increased. Changes of proportions of individual acids at the sn-3-position were compensated for by changes in the opposite direction at the sn-1and sn-2-positions. Triglyceride structure of prepartum mamma W gland secretion and early postpartum milk is examined for further knowledge of variation in positional distribution of fatty acids with changing triglyceride composition.
Triglycerides from mammary gland secretion collected at 24, 17, 10, 7, and 3.5 days prepartum and from milk collected at 0 (2 h), 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 days postpartum were subjected to fatty acid and stereospecific analysis. Prepartum mammary gland secretion differed from normal milk in that it contained approximately twice the amount of 16:0 and larger than normal amounts of 14:0. This was compensated for by less amounts of other mediumchain, short-chain, 18:0, and 18:1 acids. Positional distribution of fatty acids in milk from 1 day postpartum onward generally followed the specific pattern normally encountered for milk triglycerides. With exception of the first sample at 24 days prepartum, mammary gland secretion had medium-chain acids at 8:0, 10:0, 12:0, and sometimes 14:0 preferentially esterified at the sn-3position rather than the sn-2-position. Relationships were linear between content of fatty acids at the three stereospecific positions and content of the same acid in triglycerides. These significant relationships are represented by regression equations. With varying triglyceride fatty acid content, fatty acid content at the three positions did not change at the same rate.
MATERIALS AND METHODS Samples
INTRODUCTION
Although fatty acid composition of butter varies seasonally (6, 17), range of variation for individual fatty acids is not large. This may explain why Taylor and Hawke (23) and Received December 18, 1981.
1983 J Dairy Sci. 66:912--919
912
Secretion from mammary gland at 24, 17, 10, 7, and 3.5 days prepartum was obtained from a multiparous pregnant Friesian cow determined to be free from clinical or subclinical mastitis by the Wisconsin Mastitis Test (24). The total secretion was collected from all quarters, except at 3.5 days prepartum when only an aliquot was collected from each quarter. Lipid was extracted from the secretion by the method of Folch et al. (5). Milk samples were collected at 0 (2 h), 1, 2,
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TECHNICAL NOTE 3, 4, 5, 6; 7, 14, 21, and 28 days postpartum. Lipid was obtained by the Roese-Gottlieb method (1). Lipid was stored under nitrogen at ~20°C until required for analysis. Isolation of Triglycerides
Triglycerides were obtained from crude lipid by column chromatography with 7% hydrated Florisil (8). Stereospecific Analysis
The sn-l,2 (2,3)-diglyceride method of Brockerhoff (2), adapted for milligram quantities by Christie and Moore (3), was used with modification. This method together with the pancreatic lipase deacylation procedure used to obtain mono- and diglycerides was reported by Parodi (19). A sample of interesterified milk fat was used to obtain optimum conditions for pancreatic lipase deacylation. Fatty Acid Analysis
Glycerides and partial glycerides were transesterified to methyl esters by the method of Shehata et al. (21). Phospholipids generated during stereospecific analysis were transesterifled by the addition of 5/11 of 2.0 N methanolic sodium methoxide and 50 /al of hexane followed by shaking for 5 rain. Fatty acid methyl esters were analyzed by gas liquid chromatography (GLC) as outlined by Parodi (17, 18). RESULTS
Until 3.5 days prepartum the cow only produced about 50 ml of secretion per sampling period. Although not measured quantitatively, the secretion contained far less lipid than normal milk. Lipids from the secretion and milk were fractionated by column chromatography on Florisil to obtain the triglycerides, which were subjected to fatty acid and stereospecific analysis. Trigtyceride fatty acid composition is in Table 1. Triglycerides from prepartum mammary gland secretion differed from those of normal milk in that they contained approximately twice the 16:0 acid and larger than normal amounts of 14:0. This was compensated for by less 4:0, 6:0, 8:0, 10:0, 18:0, and 18:1 fatty acids. The positional distribution of fatty acids in triglycerides from prepartum mammary gland
~
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Journal of Dairy Science Vol. 66, No. 4, 1983
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PARODI
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sn- 1 sn-2
28[4
Sn-3
sn-1 sn-2 $n - 3
sn-1 sn-2 sn- 3 sn- i sn-2 sn-3
;;:o
7
sn-1 sn-2 $~"
P.. 14
3
sn-1 sn-2 ,¢;~ - 3
9. f~
21
< O~
Z 9
O0 ta~
28
sn- 1 sn-2 sn-3
16.5 6.1 9.7
20.2
-,3
38.7 31.3 4.2
--.7
5.4 14.3 -1,4
35.3 30.9 3.4
19.7 7.2 12.3
25.9 21.3 32.6
1.0 4.2 -.6
1.4 4.0 -1.0
6,1 14,1 -2.8
32.6 31.3 4.3
20.5 7.5 15.5
23.7 20.3 35.7
5.1
32.6 29.5 4.2
22.8 6.6 11.9
25.9 17.5 31.7
21.3 6.6 11.4
26.0 17.8 32.4
1.5 4.6 .7
16.2 3.0
.9 4.9 .5
1.7 5,1
7,2 16.5
'11:6
,2 2,4 1.6
io:2
.3 2.3 .5
.6 3.9 .0
-.4
1.1 4.1
7.7
23.0 29.8
;3:1
929
.5 2.4 .4 .4 3.4 1.0
.5 6.5 .2
,9 6.0 -.7
16.9
3i:9
.1 .1 12.7
.3 3.3 1.1
1.0 5.7 .2
1.5 5.0 -.5
16.0
;2[2
.1 .1 12.4
-.9
32.9 30.0 4.2
3i:6
,1 .1 12.6
.3 2.7 1.6
.9 5.5 .7
1.5 5.1 .5
5.9 16.3 .9
32.2 30.6 3.2
22.0 6.8 11.8
25.7 18.5 32.4
'[1
1.1 6.2 .2
1.7 5.5
12.6
,3 3.1 1.6
7.0 15.5 -2.4
30.5 29.8 3.5
19.5 6.0 15.2
24.4 17.2 39.6
.1 .2 16.7
.2 5.5 1.8
1.5 10.0 1.4
2.8 8.0 .0
8.7 19.4 .2
34.5 27.3 4.1
19.5 4.8 9.4
21.6 12.3 33.5
.1
.5 5.2 1.6
1.5 9.4
2.5 7.7
7.7 18.8
-.3
-.9
34.6 28.8 .9
19.6 4.8 8.0
22.6 12.6 39.1
26[4
sn-1 sn-2 Sn-3
20.9 19.2 30.0
.5 4.1 1,4
.3
sn-1 sn-2 SI¢l-3
13.8 5.5 8.8
,1 2,4 1,1
sn-1 sn-2
.35:6
42.9 33.5 9.7
.1 ,4 9.7
23.8
23.4
i;:8
-.8
--,9
6.1
-,9
,.q t~ :2 Z > t" Z ©
k/1
916
PAIRODI
secretion and postpartum milk is in Table 2. The distribution of individual fatty acids in milk from 1 day postpartum onward generaIly followed the specific pattern normally encountered for milk fat. The 4:0 and 6:0 fatty acids were esterified almost exclusively at the sn-3-position. The 8:0, 10:0, 12:0, and 14:0 acids were located preferentially at the sn-2 position; 16:0 was almost equally distributed between the sn-1- and sn-2-positions, and the 18:0 acid was esterified selectively at the sn-1position. The 18:1 acid usually is distributed roughly equally between the sn-1 and sn-3positions (19), but with the cow under study there was relatively less 18:1 at the sn-1position. With exception of the first sample at 24 days prepartum, mammary gland secretion had medium-chain acids 8:0, 10:0, 12:0, and sometimes 14:0 preferentially esterified at the sn-3position rather than the sn-2-position. There was also a tendency for 18:0 to be selectively esterified at the sn-2- or sn-3-positions rather than the sn-l-position. These abnormalities disappeared by the 1st day postpartum. Variation in content of individual fatty acids (10:0, 12:0, 14:0, 16:0, 18:0, and 18:1) at each of the three positions was compared with variation in total content of corresponding acids in triglycerides by regression equations. Equations were in the form y = m x + c; where y = content of fatty acid at position sn-1 or sn-2 or sn-3-; x = content of the same fatty acid in triglycerides; m and c arc constants. When c was not significantly different from zero (P<.05), the equation was in the form y=mx. Contents for 10:0 and 12:0 at the sn-2and sn-3-positions of prepartum mammary gland secretion were not included because of their abnormal distribution referred to earlier. Linear regression equations and correlation coefficients for the comparisons are in Table 3. Over the range examined, linear relationships, generally with highly significant correlation coefficients, were found between content of a fatty acid at all three positions and content of the same acid in the intact triglycerides. As the content of an acid increased in the triglycerides, there were increases in the content of that acid at the three positions. With varying triglyceride fatty acid content, the fatty acid content at Journal of Dairy Science Vol. 66, No. 4, 1983
the three positions did not change in an uniform manner. From Table 3 for the 18:1, 16:0, and 14:0 acids the change in content at the sn-3-position was more pronounced than at the sn-1- and sn-2-positions. The change in content of 18:0 was most marked at the sn-1position, and changes in 12:0 and 10:0 were more pronounced at the sn-2-position. DISCUSSION
The small quantity of mammary gland secretion produced until 3.5 days prepartum and its low content of lipid are in line with findings of Wheelock et al. (25) and Smith et al. (22), who also noted differences between animals in the amount of secretion produced. The first sample, at 24 days prepartum, had a different fatty acid composition from those at later prepartum periods. Its composition would be consistent with that of a sample of prepartum secretion diluted by milk from a previous lactation. Rowland et al. (20) found little carryover of secretion from the end of one lactation to the beginning of the next. However, little carry-over milk would have been needed to give the observed fatty acid composition. High concentrations of 16:0 and 14:0, significant amounts of 12:0 and 10:0, and low content of 18:0 in prepartum mammary gland secretion are in line with findings of Mellenberger et al. (18) and Kinsella (9). These workers measured compositions of fatty acids synthesized from [14Clacetate by mammary gland slices and homogenates obtained by biopsy from cows at various times before parturition. The other significant feature of the fatty acid composition of mammary gland secretion is the monoene fatty acids. The secretion contained higher than normal concentrations of 14:1 and 16:1 acids (confirmed by hydrogenation) and less than normal amount of 18:1. The question arises, did these acids originate from plasma triglycerides, from desaturation in the mammary gland, or from both sources? The presence of odd carbon-chain and branchedchain acids indicated plasma triglycerides were used as a source of fatty acids for milk fat synthesis. The higher contents of 14:1 and 16:1 acids corresponded to the high concentrations of 14:0 and 16:0, whereas the low content of 18:1 corresponded to the low content of 18:0. It is possible that the monoene acids resulted
TECHNICAL NOTE f r o m desaturase activity in t h e m a m m a r y gland. Kinsella and Heald (12) r e p o r t e d stearyl desaturase activity in b o v i n e m a m m a r y tissue o b t a i n e d 2 days b e f o r e p a r t u r i t i o n b u t n o t b y m a m m a r y tissue o b t a i n e d 1 to 2 wk b e f o r e p a r t u r i t i o n . T h e small a m o u n t of in vivo triglyceride s y n t h e s i s in t h e c u r r e n t s t u d y at 3.5 t o 2 4 days p r e p a r t u m w o u l d require little d e s a t u r a s e activity. This m a y n o t have b e e n d e t e c t e d in the s t u d y of Kinsella and Heald (12). A f t e r 24 d a y s p r e p a r t u m the 8:0, 10:0, and 1 2 : 0 f a t t y acids were esterified p r e f e r e n t i a l l y at t h e sn-3-position. F o l l o w i n g p a r t u r i t i o n these acids were esterified p r e f e r e n t i a l l y at t h e sn-2p o s i t i o n . T h e c o n t e n t s of these acids at the s n - 2 - p o s i t i o n were far m o r e t h a n c o n t e n t s for 37 samples of b u t t e r (19). W h e n a cow was placed on a restricted feeding regimen (19),
917
milk triglycerides b e f o r e r e s t r i c t e d feeding contained t h e 8 : 0 a n d 1 0 : 0 acids p r e f e r e n t i a l l y esterified at the sn-3-position. A f t e r r e s t r i c t e d feeding the)' were esterified p r e f e r e n t i a l l y at t h e sn-2-position. T h e s e changes do n o t a p p e a r to be related to t h e c o n t e n t of acids in the triglycerides. It is possible t h a t t h e physiological c o n d i t i o n of t h e cow m a y i n f l u e n c e some of t h e factors a f f e c t i n g a c y l a t i o n of f a t t y acids. O b t a i n i n g milk triglyceride samples e x h i b i t ing a larger t h a n n o r m a l range of f a t t y acid c o n t e n t s m a d e it possible to d e m o n s t r a t e t h e linear r e l a t i o n s h i p s b e t w e e n c o n t e n t s o f f a t t y acids at t h e t h r e e p o s i t i o n s and c o n t e n t s o f t h e same acids in the triglycerides. Christie and M o o r e (4) used 45 s a m p l e s o f pig adipose tissue w i t h a wide range of f a t t y acid c o n t e n t s and f o u n d linear r e l a t i o n s h i p s b e t w e e n p o s i t i o n a l d i s t r i b u t i o n of f a t t y acids and the a m o u n t of
TABLE 3. Linear regression analyses and correlation coefficients for the relationship between fatty acid composition at the three positions and the composition of Che same acid in the triglycerides. Fatty acid
Position
Slope
Intercept
r
18:1
sn-1 sn-2 sn- 3
1.31 .71 1.38
-9.11 . . .a . .
.
.
.970*** .649* * 824"* *
sn-1 sn-2 sn-3
1.76 .51 .95
-2.44 . . . .
. .
. .
.977"** 860"* * 901 ** *
sn-1 sn-2 sn-3
1.03 .74 1,19
10.77 13.20 - 22,94
.979* * *
14:0
sn-1 sn-2 sn-3
.66 .52 1.79
1.78 12.49 - 14.00
.967* * * .722** .949* * *
12:0
sn-1 sn-2 sn-3
.73 1.89 .36
.
sn-1 sn-2 sn-3
.38 2.40 .23
. . .
18:0
16:0
10:0
.937** * . 9 4 7 ** *
.893*** .947** * .506
"1".26 -1.21 . . .
. . .
. . .
788** 982* * * 662*
aIn these cases the intercept was not significantly different from zero, and the equation was calculated in the form y = rex. *P<.05. **P<.01. ***P<.001. Journal of Dairy Science Vol. 66, No. 4, 1983
918
PARODI
t h e same acid in the triglycerides. Litchfield (13) has t a k e n d a t a f r o m severat studies of p a n c r e a t i c Iipase d e a c y l a t i o n of seed and m a r i n e oils a n d s h o w n for some acids relationships b e t w e e n c o n t e n t s at t h e sn-2- a n d sn-l,3p o s i t i o n s a n d c o n t e n t s in triglycerides. W h e n c o n t e n t o f an acid c h a n g e d in the triglycerides, t h e c o n t e n t at the t h r e e stereospecific p o s i t i o n s did n o t change at the same rate. T h e f a t t y acid c o m p o s i t i o n of t h e sn-1 a n d s n - 2 - p o s i t i o n s is c o n t r o l l e d b y specificities of the sn-glycerol-3-phosphate and 1-acyl-sng l y c e r o l - 3 - p h o s p h a t e acyltransferases (7, 10, 14). A t t h e s n - 3 - p o s i t i o n t h e f a t t y acid c o m p o s i t i o n does n o t a p p e a r related to t h e specificities of diacylglycerol a c y l t r a n s f e r a s e s b u t r a t h e r is a f u n c t i o n of t h e c o m p o s i t i o n of t h e intracellular acyl-CoA p o o l (15). Kinsella a n d Gross (11) believe t h e r e is s o m e segregation in the m a m m a r y gland of f a t t y acids derived f r o m p l a s m a triglycerides a n d t h o s e s y n t h e s i z e d de n o v o . T h e l a t t e r m a y be utilized largely for a c y l a t i o n at t h e sn-3-position. W h e n a cow was placed o n a restricted feeding regimen (19), t h e r e was a decrease in triglycerides of t h o s e f a t t y acids s y n t h e s i z e d de novo in t h e m a m m a r y gland. T h e s e decreases were greater at t h e s n - 3 - p o s i t i o n t h a n at t h e sn-1 and s n - 2 - p o s i t i o n s a n d r e s u l t e d in triglycerides w i t h o n l y small a m o u n t s of 1 4 : 0 a n d 1 6 : 0 at t h e sn-3-position. Decreased a m o u n t s of de novo s y n t h e s i z e d f a t t y acids at t h e s n - 3 - p o s i t i o n were c o m p e n sated for b y increased c o n t e n t s of 18: 1.
ACKNOWLEDGMENTS I t h a n k Carol Beck f o r t e c h n i c a l assistance and R. E. Times, CSIRO, Division of F o o d R e s e a r c h , Dairy R e s e a r c h L a b o r a t o r y , for statistical analysis. B. E. Wilson o f the Veterin a r y Science F a r m , U n i v e r s i t y of Q u e e n s l a n d , supplied t h e m i l k samples. T h i s w o r k was supp o r t e d in p a r t b y a g r a n t f r o m t h e A u s t r a l i a n Dairy R e s e a r c h C o m m i t t e e .
REFERENCES 1 Association of Official Analytical Chemists. 1970. Official methods of analysis, l lth ed. Assoc. Offic. Anal. Chem., Washington, DC. 2 Brockerhoff, H. 1965. A stereospecific analysis of Journal of Dairy Science Vol. 66, No, 4, 1983
triglycerides. J. Lipid Res. 6:10. 3 Christie, W. W., and J. H. Moore. 1969. A Semimicro method for the stereospecific analysis of triglycerides. Biochim. Biophys. Acta 176:445. 4 Christie, W. W., and J. H. Moore. 1970. The variation of triglyceride structure with fatty acid composition in pig adipose tissue. Lipids 5:921. 5 Folch, J., M. Lees, and G.H.S. Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem.
226:497. 6 Gray, 1. K. 1973. Seasonal variations in the composition and thermal properties of New Zealand milk fat. 1. Fatty acid composition. J. Dairy Res. 40:207. 7 Gross, M. J., and J. E. Kinsella. 1974. Properties of palmityl-CoA: L-~-glycerolphosphate acyltransferase from bovine mammary mierosomes. Lipids 9:905. 8 Kates, M. 1972. Techniques of lipidology: isolation, analysis and identification of lipids. Page 269 in Laboratory techniques in biochemistry and molecular biology. T. S. Work and E. Work, ed. North Holland, Amsterdam. 9 Kinsella, J. E. 1975. Coincident synthesis of fatty acids and secretory triglycerides in bovine mammary tissue. Int. J. Biochem. 6:65. I0 Kinsella, J. E. 1976. Monoacyl-sn-glycerol-3phosphate acyltransferase specificity in bovine mammary microsomes. Lipids 11:680. 11 Kinsella, J. E., and M. Gross. 1973. Palmitic acid and initiation of mammary glyceride synthesis via phosphatidic acid. Biochim. Biophys. Acta 316: 109. 12 Kinsella, J. E., and C, W. Heald. 1972. Nal-14C stearate and Na2-14C acetate metabolism and morphological analysis of the late prepartum bovine mammary tissue. J, Dairy Sci. 55:1085. 13 Litcbfield, C. 1972. Page 233 in Analysis of triglycerides. Academic Press, New York, NY. 14 Marshall, M. O., and J. Knudsen. 1977. The specificity of 1-acyl-sn-glycerol-3-phosphate acyltransferase in microsomal fractions from lactating cow mammary gland towards short, medium, and long chain acyl-CoA esters. Biochim. Biophys. Acta 489:236. 15 Marshall, M. O., and J. Knudsen. 1979. Specificity of diacylglycerol acyltransferase from bovine mammary gland, liver and adipose tissue towards acylCoA esters. Eur. J. Biochem. 94:93. 16 Mellenberger, R. W., D. E. Bauman, and D. R. Nelson. 1973. Metabolic adaptations during lactogenesis. Fatty acid and lactose synthesis in cow mammary tissue. Biochem. J. 136:741. 17 Parodi, P. W. 1970. Fatty acid composition of Australian butter and milk fats. Australian J. Dairy Technol. 25:200. 18 Parodi, P. W. 1972. Observations on the variation in fatty acid composition of milkfat. Australian J. Dairy Technol. 27:90. 19 Parodi, P. W. 1979. Stereospecific distribution of fatty acids in bovine milk fat triglycerides. J. Dairy Res. 46:75. 20 Rowland, S. J., J.H.B. Roy, H. J. Sears, and S. Y.
TECHNICAL NOTE Thompson. 1953. The effect of prepartum milking on the composition of the prepartum and postpartum secretions of the cow. J. Dairy Res. 20:16. 21 Shehata, A. Y., J. M. de Man, and J. C. Alexander. 1970. A simple and rapid method for the preparation of methyl esters of fats in milligram amounts for gas chromatography. Can. Inst. Food Technol. J. 3:85. 22 Smith, A., J. V. Wheelock, and F. H. Dodd. 1967. Changes in the quantity and composition of mammary gland secretion in the dry period between lactations. I1. The complete dry period. J. Dairy
919
Res. 34:13. 23 Taylor, M. W., and J. C. Hawke. 1975. Structural analysis of the triacylglycerols of bovine milkfats. New Zealand J. Dairy Sci. Technol. 10:49. 24 Thompson, D. I., and D. S. Postle. 1964. The Wisconsin Mastiffs T e s t - a n indirect estimation of leucocytes in milk. J. Food Technol. 27:271. 25 Wheelock, J. V., A. Smith, F. H. Dodd, and R.L.J. Lyster. 1967. Changes in the quantity and composition of mammary gland secretion in the dry period between lactations. I. The beginning of the dry period. J. Dairy Res. 34:1.
Journal of Dairy Science Vol. 66, No. 4, 1983