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Structure and Synthesis of Milk Fat. II. Fatty Acid Distribution in the Triglycerides of Milk and Other Animal Fats1, 2
S T R U C T U R E A N D S Y N T H E S I S OF M I L K F A T . I I . F A T T Y A C I D D I S T R I B U T I O N I N T H E T R I G L Y C E R I D E S OF M I L K A N D O T H E R A N I M A L FATS*' 2 R. D. M c C A R T H Y , STUART PATTON, A~D LAURA EVAI~TS Department of Dairy Science, Pennsylvania Agricultural Experiment Station, University Park
SUMMARY Trigly~'eride structure of a number of fats was studied by use of the specificity of pancreatic lipase for cleaving the fatty acids esterified on the 1 and 3 positions of the glycerol. These fats included the back, visceral, blood, and milk triglyeerides of an individual cow; milk fat before and after seven days of inanition; visceral and back fat of a pig; and the depot fat from a steer. In all these samples the per cent of Cao, C~, and C,~ saturated acids and the C~ and C,~ monounsaturated acids, when present, were found to exist in higher concentrations in the monoglycerides resulting from the action of pancreatic lipase than in the intact fat. On the other hand, the C~ saturated and C,~ di- and triunsaturated acids were found in lower concentrations in the monoglyceride derived from all fats. In pig and milk fat C~, fatty acid was seen to be concentrated in the 2 position, while in ruminant body fats and blood triglycerides C,~ monounsaturated acid was evident at higher levels in the monoglyceride. In normal milk only a slight concentration effect of C~ acid in the 2 position was noted, indicating almost uniform distribution. However, after seven days of inanition this acid showed a high concentration in the 2-position. A comparison, based on structure and composition of blood triglycerides and milk fat in the same animal, suggests that if blood triglycerides contribute to nfilk fat there either nmst be a rearrangement of fatty acids on the glycerol or a supplementary synthesis of triglycerides which changes the pattern of fatty acid positioning. A useful method for determining glyceride structure is provided by the specificity of pancreatic lipase for hydrolyzing the ester linkages at the 1 and 3 positions of triglycerides (1, 8-10, 14, 15). This enzyme does not show a selectivity for the n a t u r e of the f a t t y acids esterified in these positions (9, 14, 15). Therefore, the f a t t y acid analysis of the original triglyceride, compared with the composition of the mouoglyccride resulting f r o m the action of pancreatic lipase, provides a measure of the f a t t y acids p r e d o m i n a n t l y esterified on the 2 position of the triglyceride. This technique was used in an extensive s t u d y b y Mattson and L u t t o n (10). They demonstrated that, with the exception of pig fat, u n s a t u r a t e d acids predominate on the 2 position of triglycerides f r o m a r a t h e r large number of animal and vegetable fats. The preferential placement of saturated acids on the 2 position of pig fat has been confirmed (13, 15). A recent r e p o r t f r o m this laboratory (11) showed t h a t milk f a t resembled pig f a t in having predominantly saturated acids on the 2 position. Since milk f a t showed positioning of f a t t y acids at variance with placement of the acids in beef f a t (10), it was deduced that at ]east two pathways of triglyceride synthesis prevail in the Received for publication April 8, 1960. ~Authorized for publication ApriI 1, 1960, :~s Paper No. 2451 in th~ Journal Series of the Pennsylvania Agricultural Experiment Station. Supported in part by the U. S. Public Health Service (It3632). 1196
STRUCTURE
AND
SYNTHESIS
OF
MILK
FAT.
11.
1197
bovine. In following this lead we analyzed the triglyceride structure of fats taken from four sources in an individual cow. These included milk, blood, visceral and back fat. In addition, milk fat, in which the fatty acid composition was modified as a result of starvation, was compared to milk from the same animal prior to withholding feed. The analyses were based on the individual fatty acids rather than the general designation of saturated and unsaturated acids. Thus, definite trends in positioning for individual acids became evident in the various fats. EXPERI1VIENTAL PROCEDURE
The four samples of fat from different locations in an individual Guernsey cow were obtained within a 2-hr. period, i.e., from last milking to slaughter. These samples included the triglycerides from milk, blood, visceral and back fat. A normal mille sample was obtained from Cow 964 before the initiation of a starvation program. At that time her daily production was 24.7 lb. of milk containing 4.1% fat. After withholding all feed for eight days her production dropped to 2.9 lb. of milk, with a fat test of 5.2%. The reported composition of milk fat produced during fasting represents the average of duplicate analyses of the A.•. milking on the sixth, seventh, and eighth day of inanition. Because of the similarity in composition the analyses have been averaged. The pig and steer fats were obtained from the meat laboratory at the University. The previous history of these animals is not precisely known. The visceral and back fat of the pig were samples from an individual animal. The steer depot fat was obtained from an animal receiving a ration designed to produce a hard or stearic-rich fat. All fat samples were extracted by essentially the same technique, using a 1:5:5 ethanol-ethyl ether-petroleum ether extraction solvent. To obtain a sufficient quantity of blood trigtyeerides, it was necessary to extract 5 liters of whole blood. Triglycerides were isolated by silicic acid chromatography (4) before being subjected to hydrolysis by pancreatic lipase according to the procedure of Mattson and Beck (8). The resulting monoglyeerides were isolated on a silicie acid column (4), their identity being confirmed by infrared spectrophotometry. After saponification the fatty acids were converted to methyl esters by refluxing 1 hr. in methanol with sulfuric acid as catalyst (3). The gas chromatographic separation of methyl esters was performed on a Barber-Colman Model 10 instrument, using a 100-ft. capillary column coated with Apiezon-L. The details of this instrumentation for separating, identifying, and quantitatively measuring fatty acid methyl esters have been reported (12). Minor component acids have not been considered in the present study nor were data taken for acids present in negligible quantities. Since the analysis of methyl but~'ate under the conditions was of questionable validity, it has not been included. The type of column used does not separate C~s diene and triene methyl esters (linoleate from linolenate) ; for this reason, they have been grouI~d together and designated in the tables as C~s"+z=.
]19~
R . D . McCARTHY, STUART PATTON, AND LAURA EVANS
RESULTS
A comparison of the results in this paper with those of Mattson and Lutton (10) shows close agreement when considered on the basis of the relative positioning of saturated and unsaturated fatty acids. Table I compares the findings for those fats common to both studies. Table 2 presents tile fatty acid composition of the original triglycerides, and the monoglycerides resulting from lipase action. In all the fats C~o, C12, C14, C1,=, and C~6= fatty acids, when present, were found to exist in higher concentrations in the monoglycerides. On the other hand, the Cls and Cls e÷s= fatty acids were found in lower coucenTABLE 1 A c o m p a r i s o n of r e s u l t s f r o m two s t u d i e s f o r p e r c e n t of t o t a l s a t u r a t e d acids in t h e 2 p o s i t i o n of f a t t r i g l y e e r i d e s Type of fat Beef Cow Cow Pig, Pig,
A, b a c k A, visceral back visceral
W e i g h t p e r c e n t of t o t a l s a t u r a t e d acids in t h e 2 position Present study 23 21 24 05 57
M a t t s o n a n d L u t t o n (10) 18 .... • 65, 67 ....
a No data.
trations in the derived monoglyeerides. In pig and milk fat, palmitic acid was seen to be concentrated in the 2 position, while in ruminant body fats a~ld blood triglycerides oleie acid was evident at higher levels in the monoglyeerides. In normal milk only a slight concentration effect of pahnitic acid in the 2 position was noted. However, after withholding feed for several days this acid had a much greater concentration in that position. The percentage of each fatty acid which was on the 2 position in the original triglycerides (Table 3) can be estimated from the composition of the triglycerides and resulting monoglycerides. With the exception of palmitic acid in normal milk, none of the fatty acids approximates random distribution. A randomly distributed acid would show a concentration of roughly 33% in the 2 position. With one exception (Cow A, visceral) the level of stearic acid in the 2 position of the trig]ycerides was rather uniform at approximately 20%. The total stearic acid content of these fats covered a range of 9 to 25%. All these fats show 25% or less of the Cls dienoic and trienoic acids in the 2 position. Regarding the placement of such acids in pig fat triglycerides, these results disagree with those of Reiser (13), who found them entirely on the 2 position. However, our results for the pig do agree with Mattson and L~tton (10), who reported an average of 22% of the linoleic and linolenie acids located on the 2 position. On the other hand, Mattson and Lutton (10) found 88% of the dienoic acid concentrated on the 2 position of beef fat, whereas our analyses show an average of 20% of the Cls dienes and trienes present in that position.
1199
STRUCTURE AND SYNTHESIS O)~ MILK FAT. II.
TABLE 2 Fatty
acid
Fatty acid
composition a of various animal f a t triglycerides a n d monoglyeerides derived t h e r e f r o m by action of p a n c r e a t i c lipase
Co
05
ClO
T y p e of f a t N o r m a l milk (Cow 964) Original trig]yceride 0.9 1.2 2.4 Monoglyceride f o r m e d < . 1 1.1 4.7 Milk, a f t e r inanition Cow (964) Original triglyceride 0.4 0.7 0.6 Monoglyceride f o r m e d < . t 0.4 1.3 Pig, visceral Original triglyceride ................ Monoglyceride f o r m e d ................ Pig, back Original triglyceride ................ Monoglyceride f o r m e d ................ Steer, depot Original triglyceride ................ Monoglyceride f o r m e d ................ Cow A, back Original triglyceride ................ Monoglyceride f o r m e d ................ Cow A, visceral Original triglyceride ........ 0.2 Monoglyceride f o r m e d ........ 0.5 Cow A, blood Original triglyceride ........ 4.5 Monoglyeeride f o r m e d ........ 7.9 Cow A, milk Original triglyceride 0.9 0.9 3.0 l~onoglyceride f o r m e d < . 1 0.8 3.8
~-d~
Ctt
2.8 5.4
12 20
016
C~6--
C18
~1~
Ol~-142-
.... b ....
38 40
1.7 2.3
14 8.7
23 15
4.0 1.9
3.9 8.0
.... ....
32 46
2.5 3.4
14 9.6
40 26
4.6 3.3
1.8 5.0
.... ....
26 60
2.8 3.5
16 8.6
42 20
12 2.7
1.7 4.6
.... ....
21 59
2.8 4.6
15 9.3
46 19
13 2.9
4.6 8.8
0.7 1.5
29 16
2.6 4.7
24 14
36 54
3.0 1.2
3.8 8.4
2.3 8.2
30 13
7.4 15
8.9 6.2
46 48
t.9 1.4
.... ....
6.1 15.0
0.8 3.5
31 18
3.2 6.8
23 8.9
34 46
1.5 1.O
.... ....
4.0 5.9
1.6 3.4
26 16
6.4 10
19 13
34 43
4.2 ....
1.2 1.4
34 39
2.1 3.2
13 9.1
27 20
3.1 1.3
1.0 1.7
2.9 4.3
12 16
CI~-
As the methyl esters a n d expressed as p e r c e n t a g e s of t o t a l c h r o m a t o g r a m area. values correspond to w e i g h t per cent. b D a t a n o t obtained.
These
DISCUSSI01~
Comparison of f at t y acid positioning on the basis of saturated and unsaturated acids as classes is not an ideal manner of determining triglyceride structure, in that it obscures patterns in the placement of the individual fat t y acids. In all the fats of this study there was a similar pattern of triglyceride structure, except in the placement of palmitic and oteic acids. I t is interesting that these fats can be differentiated on the basis of saturation or unsaturation in the 2 position, mainly because of the relative positioning of these two acids. That this is true is shown by a comparison of Tables 1 and 2. The results of this study lead us to agree with Mattson and Lutton (10), in the contention that random distribution of f a t t y acids does not occur in triglyeerides. Randomness is not compatible with 94% of the palmitic acid occurring in the 2 position of pig fat, nor does it conform with only 20% of the stearic acid being present in the 2 position of most fats. There appears to be at least a partially directed esterification for each individual fat t y acid. This is true when the results are considered either on a molar per cent or a weight per cent basis. The determining factor for this direction is not clear,
1200
R . D . ~cCARTHY, STUAI~T PATTON, AND LAURA EVANS
TABLE 3 P r o p o r t i o n s of i n d i v i d u a l f a t t y a c i d s l o c a t e d on t h e 2 p o s i t i o n i n a n u m b e r of f a t t r i g l y c e r i d e s e x p r e s s e d as p e r c e n t a g e s of t h e t o t a l c o n t e n t of t h e i n d i v i d u a l a c i d s i n t h e fats ~ Fatty acid
Co
T y p e of f a t N o r m a l m i l k (Cow 964) M i l k a f t e r i n a n i t i o n (Cow 964) Pig, visceral Pig, back Steer, d e p o t Cow A, b a c k C.ow A, v i s c e r a l Cow A, b lood Cow A, m i l k
.... b 31 65 .... 19 72 ............... ................ ................ ........ :::: .... 83 .... 59 .... 30 42
1 Calc. :
Cs
C~o
C~
C~
C~/
C16 C~ =
C,s
C~s= C~ 2÷~=
64 57
56 68 93 90 64 74 82 49 44
.... .... .... .... 71 1O0 100 71 39
34 418 77 94 18 14 19 21 38
21 23 18 21 19 23 13 23 23
22 22 16 14 50 35 4,5 42 25
.... 49
45 45 42 55 60 68 71 52 51
16 24 8 7 13 25 22 .... 14
M
× 100 = p e r cent of a c i d in 2 p o s i t i o n , w he re M is t h e p e r c e nt c o n c e n t r a t i o n 3T of t h e a c i d i n t h e m o n o g l y c e r i d e s and T is t h e p e r c e nt c o n c e n t r a t i o n of t h a t a c i d i n t h e triglycerides. b D a t a not obtained.
in that it apparently does not depend simply on chain length or degree of unsaturation. A comparison of palmitate distribution in normal milk fat, with the distribution in fat produced during inanition, raises the possibility that two mechanisms for triglyceride synthesis are operative in milk fat synthesis. In normal milk the concentration of palmitate in the 2 position was only slightly greater than the level of random distribution. However, after withholding feed from the cow for several days the concentration of palmitie acid in the 2 position became marked. This could indicate that whatever process places palmitate on the 2 position was not as greatly inhibited by starvation as the process which places this acid on the terminal positions. Of course, all mechanisms of milk fat synthesis were reduced during fasting, as shown by the substantial decrease in fat yield. Although the fatty acids of blood triglycerides have been shown to contribute to milk fat ssult.hesis (2), the quantitative contribution of this source is not known. From the present results it is apparent that if blood triglycerides contribute to milk fat, the fatty acids must either be rearranged on the glycerol molecule or there must be a supplementary synthesis of milk fat which tends to compensate by concentrating palmitate in the 2 position. The data for fatty acid composition of blood trig]ycerides from this study are essentially similar to those given in previous reports (6, 7). A comparison of fatty acid composition of blood and milk triglycerides provides a basis for interesting speculation. If it is assumed that all the oleate of milk fat comes from blood triglycerides, then the ratio of other acids to oleate in the blood triglycerides, compared with the same ratios in milk fat, indicates the blood triglycerides could provide all the oleate, linoleate, stearate, palmitoleate, myristoleate, and 50% of the palmitate plus 24% of the myristate in milk fat. In addition, the blood triglyeerides could provide all the Clo fatty acid necessary for milk fat. Only in blood and mille triglycerides was this great
STRUCTURE AND SYNTHESIS OF MILK EAT. II.
1201
a c o n c e n t r a t i o n of C,o a c i d d e t e c t e d . H o w e v e r , i n a n a l y s i s of b l o o d t r i g l y c e r i d e s f r o m a n o t h e r cow, n o t r e p o r t e d h e r e , t h e Cs, Clo, a n d C12 f a t t y a c i d s acc o u n t e d f o r 3 % o f t h e t o t a l f a t t y acids. T i l e p r e s e n c e o f s h o r t c h a i n a c i d s i n blood may have significance in relation to the recent report of James and L o v e l o e k ( 5 ) , on t h e f a t t y a c i d s y n t h e s i z i n g a b i l i t y of w h o l e b l o o d .
REFEI~ENCES
(1) BORGSTRO~, B. On tile Mechanism of Pancreatic Lipolysis of G]ycerides. Bioehim. et Biophys. Acta, 13: 491. 1954. (2) GLASCOCK, ]~. F. Recent Research on the Origin of Milk Fat. Prec. Roy. Soe., 149: 402. 1958. (3) I4ILI)ITCH, T. P. The Chemical Constitution of Natural Fats. 3rd ed. p. 575. John Wiley and Sons, New York. 1956. (4) Hmscri, J., AND AI4RENS, E. M. The Separation of Complex Lipide Mixtures by the Use of Silicic Acid Chromatography. J. Biol. Chem., 233': 311. 1958. (5) JAM~S, A. T., AN]) LOVE~)CK, J. E. The Lipids of Whole Blood. 1. Lipid Biosynthesis in Human Blood in Vitro. Biochem. J., 73: 106. 1959. (6) ~KE~S~Y, F. E., AND LONGE~ECKE% H. E. Distribution and Characterization of Beef Plasma Fatk~ Acids. J. Biol. Chem., 139 : 727. 1941. (7) LOUGH, A. K., AN]) GA~TON, G. A. Blood Lipids. 2. Plasma Lipids of the Lactating Cow. F a t t y Acid Composition of Sterol Esters and Trlglyeerldes. Biochem. J., 67: 345. 1957. (8) M~TTSON, F. I-L, AN]) BE CE, L. W. The Digestion in Vitro of Triglycerides by Pancreatic Lipase. J. Biol. Chem., 214: 115. 1955. (9) MA~TSON, F. H., AN]) BECK, L. W. The Specificity of Pancreatic Lipase for the Primary Hydroxyl Groups of Glycerides. J. Biol. Chem., 219:735. 1956. (10) MATTSO~, F. H., AND LYTTON, E. S. The Specific Distribution of 'Fatty Acids in the Glyccrides of Animal and Vegetable Fats. J. Biol. Chem., 233:860. 1958. (11) P.~TTON, Sa~TART,EVANS, LAURA, A~D 5'[cCAI~T~t¥, R. D. The Action of Pancreatic Lipase on Milk Fat. J. Dairy Sci., 43: 95. 1960. (]2) PATTON, STUA!~T, MCCARTHY, ]~. D., EVAiN-S, LAUR.A~AND LYNN~ R. T. Structure and Synthesis of Milk Fat. I. Gas Chromatographic Analysis. J. Dairy Scl, 43 : 1187. 1960. (13) RSlS~, R., AN]) R~IAKSISHMA REDDY, H. G. The Glyceride Structure of Swine Depot Fat. J. Am. Oil Chemists' Soe., 36: 97. 1959. (].4) SAVAICY,P., AN])DESNU]~LL~ P. Sur quelques Elements de Specificite pendant l'Hydrolyse enzymat~que des Triglycerides. Bioehim. et Biophys. Aeta, 21: 349. 1956. (15) ¥O[~NGS, C. G. Glycerlde Structure of Fats. J. Am. Oil Chemists' Soc., 36: 664. ]959.
SUMMARY Trigly~'eride structure of a number of fats was studied by use of the specificity of pancreatic lipase for cleaving the fatty acids esterified on the 1 and 3 positions of the glycerol. These fats included the back, visceral, blood, and milk triglyeerides of an individual cow; milk fat before and after seven days of inanition; visceral and back fat of a pig; and the depot fat from a steer. In all these samples the per cent of Cao, C~, and C,~ saturated acids and the C~ and C,~ monounsaturated acids, when present, were found to exist in higher concentrations in the monoglycerides resulting from the action of pancreatic lipase than in the intact fat. On the other hand, the C~ saturated and C,~ di- and triunsaturated acids were found in lower concentrations in the monoglyceride derived from all fats. In pig and milk fat C~, fatty acid was seen to be concentrated in the 2 position, while in ruminant body fats and blood triglycerides C,~ monounsaturated acid was evident at higher levels in the monoglyceride. In normal milk only a slight concentration effect of C~ acid in the 2 position was noted, indicating almost uniform distribution. However, after seven days of inanition this acid showed a high concentration in the 2-position. A comparison, based on structure and composition of blood triglycerides and milk fat in the same animal, suggests that if blood triglycerides contribute to nfilk fat there either nmst be a rearrangement of fatty acids on the glycerol or a supplementary synthesis of triglycerides which changes the pattern of fatty acid positioning. A useful method for determining glyceride structure is provided by the specificity of pancreatic lipase for hydrolyzing the ester linkages at the 1 and 3 positions of triglycerides (1, 8-10, 14, 15). This enzyme does not show a selectivity for the n a t u r e of the f a t t y acids esterified in these positions (9, 14, 15). Therefore, the f a t t y acid analysis of the original triglyceride, compared with the composition of the mouoglyccride resulting f r o m the action of pancreatic lipase, provides a measure of the f a t t y acids p r e d o m i n a n t l y esterified on the 2 position of the triglyceride. This technique was used in an extensive s t u d y b y Mattson and L u t t o n (10). They demonstrated that, with the exception of pig fat, u n s a t u r a t e d acids predominate on the 2 position of triglycerides f r o m a r a t h e r large number of animal and vegetable fats. The preferential placement of saturated acids on the 2 position of pig fat has been confirmed (13, 15). A recent r e p o r t f r o m this laboratory (11) showed t h a t milk f a t resembled pig f a t in having predominantly saturated acids on the 2 position. Since milk f a t showed positioning of f a t t y acids at variance with placement of the acids in beef f a t (10), it was deduced that at ]east two pathways of triglyceride synthesis prevail in the Received for publication April 8, 1960. ~Authorized for publication ApriI 1, 1960, :~s Paper No. 2451 in th~ Journal Series of the Pennsylvania Agricultural Experiment Station. Supported in part by the U. S. Public Health Service (It3632). 1196
STRUCTURE
AND
SYNTHESIS
OF
MILK
FAT.
11.
1197
bovine. In following this lead we analyzed the triglyceride structure of fats taken from four sources in an individual cow. These included milk, blood, visceral and back fat. In addition, milk fat, in which the fatty acid composition was modified as a result of starvation, was compared to milk from the same animal prior to withholding feed. The analyses were based on the individual fatty acids rather than the general designation of saturated and unsaturated acids. Thus, definite trends in positioning for individual acids became evident in the various fats. EXPERI1VIENTAL PROCEDURE
The four samples of fat from different locations in an individual Guernsey cow were obtained within a 2-hr. period, i.e., from last milking to slaughter. These samples included the triglycerides from milk, blood, visceral and back fat. A normal mille sample was obtained from Cow 964 before the initiation of a starvation program. At that time her daily production was 24.7 lb. of milk containing 4.1% fat. After withholding all feed for eight days her production dropped to 2.9 lb. of milk, with a fat test of 5.2%. The reported composition of milk fat produced during fasting represents the average of duplicate analyses of the A.•. milking on the sixth, seventh, and eighth day of inanition. Because of the similarity in composition the analyses have been averaged. The pig and steer fats were obtained from the meat laboratory at the University. The previous history of these animals is not precisely known. The visceral and back fat of the pig were samples from an individual animal. The steer depot fat was obtained from an animal receiving a ration designed to produce a hard or stearic-rich fat. All fat samples were extracted by essentially the same technique, using a 1:5:5 ethanol-ethyl ether-petroleum ether extraction solvent. To obtain a sufficient quantity of blood trigtyeerides, it was necessary to extract 5 liters of whole blood. Triglycerides were isolated by silicic acid chromatography (4) before being subjected to hydrolysis by pancreatic lipase according to the procedure of Mattson and Beck (8). The resulting monoglyeerides were isolated on a silicie acid column (4), their identity being confirmed by infrared spectrophotometry. After saponification the fatty acids were converted to methyl esters by refluxing 1 hr. in methanol with sulfuric acid as catalyst (3). The gas chromatographic separation of methyl esters was performed on a Barber-Colman Model 10 instrument, using a 100-ft. capillary column coated with Apiezon-L. The details of this instrumentation for separating, identifying, and quantitatively measuring fatty acid methyl esters have been reported (12). Minor component acids have not been considered in the present study nor were data taken for acids present in negligible quantities. Since the analysis of methyl but~'ate under the conditions was of questionable validity, it has not been included. The type of column used does not separate C~s diene and triene methyl esters (linoleate from linolenate) ; for this reason, they have been grouI~d together and designated in the tables as C~s"+z=.
]19~
R . D . McCARTHY, STUART PATTON, AND LAURA EVANS
RESULTS
A comparison of the results in this paper with those of Mattson and Lutton (10) shows close agreement when considered on the basis of the relative positioning of saturated and unsaturated fatty acids. Table I compares the findings for those fats common to both studies. Table 2 presents tile fatty acid composition of the original triglycerides, and the monoglycerides resulting from lipase action. In all the fats C~o, C12, C14, C1,=, and C~6= fatty acids, when present, were found to exist in higher concentrations in the monoglycerides. On the other hand, the Cls and Cls e÷s= fatty acids were found in lower coucenTABLE 1 A c o m p a r i s o n of r e s u l t s f r o m two s t u d i e s f o r p e r c e n t of t o t a l s a t u r a t e d acids in t h e 2 p o s i t i o n of f a t t r i g l y e e r i d e s Type of fat Beef Cow Cow Pig, Pig,
A, b a c k A, visceral back visceral
W e i g h t p e r c e n t of t o t a l s a t u r a t e d acids in t h e 2 position Present study 23 21 24 05 57
M a t t s o n a n d L u t t o n (10) 18 .... • 65, 67 ....
a No data.
trations in the derived monoglyeerides. In pig and milk fat, palmitic acid was seen to be concentrated in the 2 position, while in ruminant body fats a~ld blood triglycerides oleie acid was evident at higher levels in the monoglyeerides. In normal milk only a slight concentration effect of pahnitic acid in the 2 position was noted. However, after withholding feed for several days this acid had a much greater concentration in that position. The percentage of each fatty acid which was on the 2 position in the original triglycerides (Table 3) can be estimated from the composition of the triglycerides and resulting monoglycerides. With the exception of palmitic acid in normal milk, none of the fatty acids approximates random distribution. A randomly distributed acid would show a concentration of roughly 33% in the 2 position. With one exception (Cow A, visceral) the level of stearic acid in the 2 position of the trig]ycerides was rather uniform at approximately 20%. The total stearic acid content of these fats covered a range of 9 to 25%. All these fats show 25% or less of the Cls dienoic and trienoic acids in the 2 position. Regarding the placement of such acids in pig fat triglycerides, these results disagree with those of Reiser (13), who found them entirely on the 2 position. However, our results for the pig do agree with Mattson and L~tton (10), who reported an average of 22% of the linoleic and linolenie acids located on the 2 position. On the other hand, Mattson and Lutton (10) found 88% of the dienoic acid concentrated on the 2 position of beef fat, whereas our analyses show an average of 20% of the Cls dienes and trienes present in that position.
1199
STRUCTURE AND SYNTHESIS O)~ MILK FAT. II.
TABLE 2 Fatty
acid
Fatty acid
composition a of various animal f a t triglycerides a n d monoglyeerides derived t h e r e f r o m by action of p a n c r e a t i c lipase
Co
05
ClO
T y p e of f a t N o r m a l milk (Cow 964) Original trig]yceride 0.9 1.2 2.4 Monoglyceride f o r m e d < . 1 1.1 4.7 Milk, a f t e r inanition Cow (964) Original triglyceride 0.4 0.7 0.6 Monoglyceride f o r m e d < . t 0.4 1.3 Pig, visceral Original triglyceride ................ Monoglyceride f o r m e d ................ Pig, back Original triglyceride ................ Monoglyceride f o r m e d ................ Steer, depot Original triglyceride ................ Monoglyceride f o r m e d ................ Cow A, back Original triglyceride ................ Monoglyceride f o r m e d ................ Cow A, visceral Original triglyceride ........ 0.2 Monoglyceride f o r m e d ........ 0.5 Cow A, blood Original triglyceride ........ 4.5 Monoglyeeride f o r m e d ........ 7.9 Cow A, milk Original triglyceride 0.9 0.9 3.0 l~onoglyceride f o r m e d < . 1 0.8 3.8
~-d~
Ctt
2.8 5.4
12 20
016
C~6--
C18
~1~
Ol~-142-
.... b ....
38 40
1.7 2.3
14 8.7
23 15
4.0 1.9
3.9 8.0
.... ....
32 46
2.5 3.4
14 9.6
40 26
4.6 3.3
1.8 5.0
.... ....
26 60
2.8 3.5
16 8.6
42 20
12 2.7
1.7 4.6
.... ....
21 59
2.8 4.6
15 9.3
46 19
13 2.9
4.6 8.8
0.7 1.5
29 16
2.6 4.7
24 14
36 54
3.0 1.2
3.8 8.4
2.3 8.2
30 13
7.4 15
8.9 6.2
46 48
t.9 1.4
.... ....
6.1 15.0
0.8 3.5
31 18
3.2 6.8
23 8.9
34 46
1.5 1.O
.... ....
4.0 5.9
1.6 3.4
26 16
6.4 10
19 13
34 43
4.2 ....
1.2 1.4
34 39
2.1 3.2
13 9.1
27 20
3.1 1.3
1.0 1.7
2.9 4.3
12 16
CI~-
As the methyl esters a n d expressed as p e r c e n t a g e s of t o t a l c h r o m a t o g r a m area. values correspond to w e i g h t per cent. b D a t a n o t obtained.
These
DISCUSSI01~
Comparison of f at t y acid positioning on the basis of saturated and unsaturated acids as classes is not an ideal manner of determining triglyceride structure, in that it obscures patterns in the placement of the individual fat t y acids. In all the fats of this study there was a similar pattern of triglyceride structure, except in the placement of palmitic and oteic acids. I t is interesting that these fats can be differentiated on the basis of saturation or unsaturation in the 2 position, mainly because of the relative positioning of these two acids. That this is true is shown by a comparison of Tables 1 and 2. The results of this study lead us to agree with Mattson and Lutton (10), in the contention that random distribution of f a t t y acids does not occur in triglyeerides. Randomness is not compatible with 94% of the palmitic acid occurring in the 2 position of pig fat, nor does it conform with only 20% of the stearic acid being present in the 2 position of most fats. There appears to be at least a partially directed esterification for each individual fat t y acid. This is true when the results are considered either on a molar per cent or a weight per cent basis. The determining factor for this direction is not clear,
1200
R . D . ~cCARTHY, STUAI~T PATTON, AND LAURA EVANS
TABLE 3 P r o p o r t i o n s of i n d i v i d u a l f a t t y a c i d s l o c a t e d on t h e 2 p o s i t i o n i n a n u m b e r of f a t t r i g l y c e r i d e s e x p r e s s e d as p e r c e n t a g e s of t h e t o t a l c o n t e n t of t h e i n d i v i d u a l a c i d s i n t h e fats ~ Fatty acid
Co
T y p e of f a t N o r m a l m i l k (Cow 964) M i l k a f t e r i n a n i t i o n (Cow 964) Pig, visceral Pig, back Steer, d e p o t Cow A, b a c k C.ow A, v i s c e r a l Cow A, b lood Cow A, m i l k
.... b 31 65 .... 19 72 ............... ................ ................ ........ :::: .... 83 .... 59 .... 30 42
1 Calc. :
Cs
C~o
C~
C~
C~/
C16 C~ =
C,s
C~s= C~ 2÷~=
64 57
56 68 93 90 64 74 82 49 44
.... .... .... .... 71 1O0 100 71 39
34 418 77 94 18 14 19 21 38
21 23 18 21 19 23 13 23 23
22 22 16 14 50 35 4,5 42 25
.... 49
45 45 42 55 60 68 71 52 51
16 24 8 7 13 25 22 .... 14
M
× 100 = p e r cent of a c i d in 2 p o s i t i o n , w he re M is t h e p e r c e nt c o n c e n t r a t i o n 3T of t h e a c i d i n t h e m o n o g l y c e r i d e s and T is t h e p e r c e nt c o n c e n t r a t i o n of t h a t a c i d i n t h e triglycerides. b D a t a not obtained.
in that it apparently does not depend simply on chain length or degree of unsaturation. A comparison of palmitate distribution in normal milk fat, with the distribution in fat produced during inanition, raises the possibility that two mechanisms for triglyceride synthesis are operative in milk fat synthesis. In normal milk the concentration of palmitate in the 2 position was only slightly greater than the level of random distribution. However, after withholding feed from the cow for several days the concentration of palmitie acid in the 2 position became marked. This could indicate that whatever process places palmitate on the 2 position was not as greatly inhibited by starvation as the process which places this acid on the terminal positions. Of course, all mechanisms of milk fat synthesis were reduced during fasting, as shown by the substantial decrease in fat yield. Although the fatty acids of blood triglycerides have been shown to contribute to milk fat ssult.hesis (2), the quantitative contribution of this source is not known. From the present results it is apparent that if blood triglycerides contribute to milk fat, the fatty acids must either be rearranged on the glycerol molecule or there must be a supplementary synthesis of milk fat which tends to compensate by concentrating palmitate in the 2 position. The data for fatty acid composition of blood trig]ycerides from this study are essentially similar to those given in previous reports (6, 7). A comparison of fatty acid composition of blood and milk triglycerides provides a basis for interesting speculation. If it is assumed that all the oleate of milk fat comes from blood triglycerides, then the ratio of other acids to oleate in the blood triglycerides, compared with the same ratios in milk fat, indicates the blood triglycerides could provide all the oleate, linoleate, stearate, palmitoleate, myristoleate, and 50% of the palmitate plus 24% of the myristate in milk fat. In addition, the blood triglyeerides could provide all the Clo fatty acid necessary for milk fat. Only in blood and mille triglycerides was this great
STRUCTURE AND SYNTHESIS OF MILK EAT. II.
1201
a c o n c e n t r a t i o n of C,o a c i d d e t e c t e d . H o w e v e r , i n a n a l y s i s of b l o o d t r i g l y c e r i d e s f r o m a n o t h e r cow, n o t r e p o r t e d h e r e , t h e Cs, Clo, a n d C12 f a t t y a c i d s acc o u n t e d f o r 3 % o f t h e t o t a l f a t t y acids. T i l e p r e s e n c e o f s h o r t c h a i n a c i d s i n blood may have significance in relation to the recent report of James and L o v e l o e k ( 5 ) , on t h e f a t t y a c i d s y n t h e s i z i n g a b i l i t y of w h o l e b l o o d .
REFEI~ENCES
(1) BORGSTRO~, B. On tile Mechanism of Pancreatic Lipolysis of G]ycerides. Bioehim. et Biophys. Acta, 13: 491. 1954. (2) GLASCOCK, ]~. F. Recent Research on the Origin of Milk Fat. Prec. Roy. Soe., 149: 402. 1958. (3) I4ILI)ITCH, T. P. The Chemical Constitution of Natural Fats. 3rd ed. p. 575. John Wiley and Sons, New York. 1956. (4) Hmscri, J., AND AI4RENS, E. M. The Separation of Complex Lipide Mixtures by the Use of Silicic Acid Chromatography. J. Biol. Chem., 233': 311. 1958. (5) JAM~S, A. T., AN]) LOVE~)CK, J. E. The Lipids of Whole Blood. 1. Lipid Biosynthesis in Human Blood in Vitro. Biochem. J., 73: 106. 1959. (6) ~KE~S~Y, F. E., AND LONGE~ECKE% H. E. Distribution and Characterization of Beef Plasma Fatk~ Acids. J. Biol. Chem., 139 : 727. 1941. (7) LOUGH, A. K., AN]) GA~TON, G. A. Blood Lipids. 2. Plasma Lipids of the Lactating Cow. F a t t y Acid Composition of Sterol Esters and Trlglyeerldes. Biochem. J., 67: 345. 1957. (8) M~TTSON, F. I-L, AN]) BE CE, L. W. The Digestion in Vitro of Triglycerides by Pancreatic Lipase. J. Biol. Chem., 214: 115. 1955. (9) MA~TSON, F. H., AN]) BECK, L. W. The Specificity of Pancreatic Lipase for the Primary Hydroxyl Groups of Glycerides. J. Biol. Chem., 219:735. 1956. (10) MATTSO~, F. H., AND LYTTON, E. S. The Specific Distribution of 'Fatty Acids in the Glyccrides of Animal and Vegetable Fats. J. Biol. Chem., 233:860. 1958. (11) P.~TTON, Sa~TART,EVANS, LAURA, A~D 5'[cCAI~T~t¥, R. D. The Action of Pancreatic Lipase on Milk Fat. J. Dairy Sci., 43: 95. 1960. (]2) PATTON, STUA!~T, MCCARTHY, ]~. D., EVAiN-S, LAUR.A~AND LYNN~ R. T. Structure and Synthesis of Milk Fat. I. Gas Chromatographic Analysis. J. Dairy Scl, 43 : 1187. 1960. (13) RSlS~, R., AN]) R~IAKSISHMA REDDY, H. G. The Glyceride Structure of Swine Depot Fat. J. Am. Oil Chemists' Soe., 36: 97. 1959. (].4) SAVAICY,P., AN])DESNU]~LL~ P. Sur quelques Elements de Specificite pendant l'Hydrolyse enzymat~que des Triglycerides. Bioehim. et Biophys. Aeta, 21: 349. 1956. (15) ¥O[~NGS, C. G. Glycerlde Structure of Fats. J. Am. Oil Chemists' Soc., 36: 664. ]959.