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Pancreatic Lipase Hydrolysis of Cow Milk Fat1
PANCREATIC E. L. JACK,
LIPASE HYDROLYSIS OF COW MILK FAT 1 C. P. FREEMAN,
L. M.
SMITH,
AN]) J. B. MICKLE
~
Department of Food Science and Technology, University of California, Davis SUMMARY
Knowledge of the position of individual fatty acids within the trig'lycerides is necessary to the understanding of the utilization of a fat. Pancreatic lipase hydrolysis to convert triglycerides to 2-monoglycerides has been used to study this type of glyceride structure in roany food fats, but it has recently been claimed that it cannot be used with cow milk fat because butyric acid is hydrolyzed more rapidly than the other acids. This study was undertaken to determine if there were conditions under which this technique could be applied validly to milk fat. The criteria set forth for the applicability of this technique were: (a) nonpreferential hydrolysis of triglyceride species; (b) absence of a substantial amount of complete hydrolysis; and (c) absence of a significant amount of acyl migration during the hydrolysis reaction. Although there was no evidence of preferential hydrolysis at the one-third stage, there was some complete hydrolysis, and acyl migration did not appear to be occurring at a significant rate. The validity of the procedures employed was demonstrated on a fat of known and unique structure, pig body fat. Therefore, the technique can give ~ s u l t s that may be used to establish general relationships. I n cow milk fat the majority of the fatty acids were found to be uniformly distributed within the glyceride, except for C, and C~, which are predominantly in the external positions, and C~, which tends to concentrate in the two position.
Knowledge of the position of the individual fatty acids within the triglycerides is important to the understanding of the synthesis, digestion and metabolism, and technology of fat. Recent developments in analytical techniques have made possible the acquisition of this knowledge for a nmnber of fats (1, 11, 12, 16, 19). The key technique in such studies is the application of controlled lipolysis to convert triglyeerides to monoglycerides. I t has been known for some time that pancreatic lipase hydrolyzes triglycerides to partial glycerides, but it remained for Mattson and Beck (10) and Savary et al. (15) to demonstrate that the action of the enzyme is primarily on the terminal fatty acid groups, and that the monoglycerides resulting are predominantly 2-monoglyeerides. Thus, by determining the fatty acids in the nmnoglyeerides, the position of the fatty acids in the triglyeerides is revealed. Other reports have confirmed the specificity of pancreatic and also nfilk lipases for the terminal acid groups (2, 6, 17). Received for publication December 17, 1962. Supported in part by PHS Research Grant A4250 from National Institute of Physiological Chemistry, Public Health Service. Postdoctoral fellow on sabbatical leave from Oklahoma State University, Stillwater, Oklahoma, 1961.
I n general, studies using this technique have shown that there is a nonrandom distribution of some of the fatty acids within the triglycerides, with the unsaturated fatty acids tending to be characteristically in the two position except for pig body fat, where they are in the one or three positions. Studies on cow milk fat using pancreatic lipase hydrolysis have not been sufficiently comprehensive for firm conclusions to be drawn. McCarthy et al. (12) reported results on milk fat produced from cows with normal feeding and after inanition, and related the composition to that of blood and body fat. They found that myristic acid (C1~) tends to concentrate in the two position and the C~ unsaturated acids in the external positions. Thus, from these data cow nfilk fat resembles lard in this respect. However, these investigators did not determine the lower fatty acids--C, and Co--and it is difficult to draw over-all conclusions. The same statement applies to the results by Ast and Vander Wal (1). Their conclusions are in agreement with those of McCarthy et al., but again the data for the lower fatty acids are questionable or lacking. A short paper by K u m a r et al. (8) deals with butyric acid (C,), which they find to be located ahnost exclusively in the one or three positions. Desnuelle's group, Entressangles et al. (5), has criticized this latter paper as the result of 284
LIPASE
HYDROLYSIS
their investigation of the effect of fatty acid chain length on the rate of hydrolysis by pancreatic lipase. They show that the short-chain acids are hydrolyzed nmre rapidly than the long-chain and that C~ is hydrolyzed the most rapidly of all. They state that it is thus impossible, as K u m a r et a l. (8) have claimed, to deternfine the position of the short-chain acids in the glyeerides of milk fat with a technique based on the positional specificity of lipase. Because this technique is being widely used in the study of glyceride structure, and its use is being extended to milk fat., where it offers a method of establishing the glyeeride structure of this complex fat, it becomes necessary to determine the validity of this statement. I t is a common observation, as documented by E'ntressangles et al. (5), that butyric acid is hydrolyzed more rapidly than the longerchain acids in both simple and nfixed glycerides, and that there is some difference in rates among fatty acids generally, depending upon chain length and other factors. However, since the monoglyceride formed is the critical compound in this analysis, the essential point is whether the monogIyceride is representative of the fat as a whole. I t is our belief that if the hydrolysis reaction is carried out in such a way that: (a) there is no preferential hydrolysis of any partitular triglyceride species, (b) a substantial amount of complete hydrolysis of the glycerides does not occur, and- (c) there is no appreciable acyl migration from the two position during the course of the reactions; then, under these conditions, the resulting monoglycerides are a reliable index of the positional coufigur3.tion of the fat. Experiments to test these criteria of the applicability of pancreatic lipase hydrolysis to cow milk fat are reported. EXPERIlVIENTAL PROCEDURE
I t seemed desirable to work initially with known materials in establishing whether the conditions set forth above could be attained. A nlixture of triglycerides containing butyric, caproic, caprylic, laurie, myristic, pahnitie, stearie, and oleic acids was chosen to simulate milk fat in range of fatty acid chain length and in amounts. Hydrolysis of this mixture as simple triglyeerides using pancreatic lipase was not satisfactory, because the long-chain group was not liquid at the temperature of hydrolysis, thus reducing greatly, or inhibiting entirely, its hydrolysis. Consequently, the nfixture was interesterified and satisfactory perforlnance was obtained. From prelinfinary
OF
MILK
FAT
285
trials with this interesterified glyceride mixture, conditions of hydrolysis were established which promised to be reliably applicable to inilk fat. Cow milk fat was then analyzed by the following procedures: After separating the triglyeerides from the crude fat by column chromatography, these were digested with pancreatie lipase to an appropriate stage as established by the preliminary trials, the digested mixture was separated into the free fatty acids and the different glyceride classes by colmnn chromatography, and the acids from the individual groups were then methylated and analyzed by GLC. To confirm the validity of the procedures, pig body fat, which has been thoroughly studied and which has been shown to have a characteristic positional patting, was similarly mlalyzed. Methods. Preparation of the fats. Cow milk fat was obtained by churning cream and melting and filtering the fat according to the procedure described by Smith (18). The pig body fat was taken from raw pork obtained locally. The back fat was homogenized 3-5 rain at room temperature in a Waring Blendor with 20 vol of CH~C1-MeOH (2:1) as solvent per gram of fat. The mixture was fltered through sintered glass and the solvent removed under reduced pressure. The dried lipid was re-extracted with 10 vol of CH~C1-MeOII solvent. The triglycerides from both the milk fat and the pig fat were separated from other materials as follows: Two grams of fat was taken up in a small volume o~ hexane and applied on to a colunto (140 g; 56 X 2.5 cm) of Florisil which had been oven-dried at 110 C for 24 hr. The column was eluted successively with 250 nfl of hexane, 1,500 ml of 50% ether in hexale, and 1,000 ml of 75% ether in hexane. The first 400 ml of eluate was discarded, and the next 1,000 nil, containing the triglycerides, was collected in bulk. Subsequent eluate was collected in 200-ml fractions and exanfined by thin-la~er chromatography to insure that the triglycerides were completely recovered from the column, and to check whether diglyceride was beginning to appear in the eluate. Those fractions containing triglycerides were added to the bulk, and the solvent removed at 0 C under reduced pressure. Pancreatic lipase digestion. The digestion nfixture contained 2.0 lnl of 45% CaCI~, 25 ml of 1.0 ~ NH~C1, and 10 ml of a 4% aqueous suspension of ground pork pancrease ~ which h3d been extracted previously with ethyl ether. 3 Mann Research Laboratories. Inc., New York
6, N . Y .
286
E.L. J~OK ~T ~L
Concentrated NH,OH was added to bring the p H to 8.0. This mixture was diluted to 100 ml with H~O, warmed to 40 C, and added to 0.1-0.3 g of triglyceride substrate. Digestion was carried out at 40 C with constant stirring and addition of NH~OI{ to maintain the p i t at 7.8-8.2. The reaction was stopped at an a p p r o p r i a t e stage by acidifying to p i t 2.0 with HC1. The glycerides and f a t t y acids were then extracted three times with 300 ml of ethyl ether-hexane (1:1). This extraction procedure recovered over 90% of the butyric acid and 100% of the other acids and glycerides. Separation of glyceride cl,asses. The silicic acid or silica gel eolunms, as described by Itirsch and Ahrens (7) or by Quinlin and Weiser (13), have been used most conmmnly to separate glyceride classes. W i t h milk fat, we were unable to get consistently satisfactorily distinct and quantitative separations with these procedures. However, the Florisil column technique of Carroll (4) was modified to give satisfactory results. The following procedures were adopted: Florisil was oven-dried at 110 C for 24 hr and 12 g was packed as a slurry in hexane in a colmnn (26 × 1 cm). The load was 100-150 mg. The eluting solvents were 250 ml of 60% ethyl ether in hexane, followed by 200 ml of 0.35% methanol in ethyl ether, then by 150 ml of 5.0% methanol in ethyl ether, and finally by 100 ml of 4% acetic acid in ethyl ether. These solvents remove tri-, di-, and monoglycerides, and f a t t y acids, in that order. Figure 1 shows the sharpness of separation of the different glyceride groups in a nfilk fat hydrolysate. Mcthylation and GLC. A f t e r the extracted lipids wer e separated into classes by the column chronmtographic procedure described above, the f a t t y acids front each glyceride class were methi
i
i
~ 6 o % ETHERIN HEXANE
,1~0.35~/o MeOHIN ETHER11--~5% MeOHtN ETHER~ I I
1 I I
F
D~-
TRY-
1 MONOGLYCERIDES
i I
I i
~0
I0
20
_
30 FRACTION
40
50
60
FrG. 1. Separation of glycerides i~t ~ milk fat hydrolysat~ on Florisil c o l u m n .
ylated and analyzed by GLC as described by Smith (18). RESULTS
Experiments with cow milk fat. Evidence bearing on perferential hydrolysis. In pancreatic lipase hydrolysis, the digestion is usually stopped when some fraction of the total f a t t y acids has been hydrolyzed. F r e quently, this is at about 60% hydrolysis. Among the residual materials are free fatty acids, mono-, di-, and triglyeerides. I f the enzyme has not preferentially attacked any pal~icular triglyeeride species, the residual triglycerides should resemble closely in composition the triglycerides from the original fat. PreliminaIT trials with the interesterified mixture showed this condition to obtain when 30-40% of the fatty acids had been hydrolyzed. Consequently, milk f a t was hydro]yzed and analyzed as described earlier. Digestions were carried to the one-third and two-thirds stages for comparison. Table 1 shows the results. The agreement between the composition of the triglycer{des of the original fat and that of the residual triglyeerides is remarkably close at one-third hydrolysis, indicating that at this stage there has not been significant preferential hydrolysis among the f a t t y acids at the terminal positions. A t the two-thirds stage the agreement is not quite as good, particularly with butyric acid. Rate of attainment of complete hydrolysis. A satisfactmT measure of complete hydrolysis is the appearance of free glycerol. Consequently, measurements of the amount of free glycerol were made by the method of Lambeth and Neish (9) at different stages in the digestion process. The prelimina~T trials with interesterified glycerides showed that only small amounts of free glycerol appeared up to 3040% hydrolysis. Accordingly, determinations of free glycerol were made at one-third and two-thirds hydrolysis. Table 2 shows the results obtained on both the milk fat and the pig fat. Assuming the average chain length of the f a t t y acids in milk fat to be approximately 14 carbon atoms, the amount of free glycerol produced at one-third hydrolysis is about 8% of the total glycerol. The molaz" amounts of free glycerol for pig fat at one-third hydrolysis are approximately 2-3% and 11-12% at two-thirds hydrolysis. E~idence bearing on acyl migration. Migration of f a t t y acids among the positions within glycerides is known to occur under certain conditions and if the acids in the two position migrate to the external positions in any appre-
LIPASE
HYDROLYSIS
TABLE
OF
MILK
FAT
287
1
Composition of cow milk fat and of residual glycerides after pancreatic lipase hydrolysis l~atty acid chain length 4 6 8 10 10 : 1 12 12:1 13 14 br. 14 14:1
15 16 br. 16 16:1 17 br.
Residual glyeerides OriginM fat 11.1 5.4 2.1 4.4 0.4 4.1 (}.2 0.1 0.1 11.7 1.5 1.3 0.1 23.7 2.6 0.6
17 18 br. 18
0.5 0.3 8.8
18:1
18.6
18:2 I8:3
1.6 0.8
Hydrolysis Tri
Di
Pig fat--200 mg
T1d
Di
Mono
5.9 4.1
1:6
9.7 5.2
9.3 5.1
2.0
1.o
1.0
1.3
1.4
0.6
4.5 0.6 4.1 0.3 0.3 0.1 11.8 1.8 1.1 0.1 24.4 2.9 0'N 0.2
4.6 0.5 4.~ 0.2 0.1 0.~ 14.0 2.0 1.5 0.5 2'4.4 3.6 0.3 0.1 0.2 6.9 17.3 2.2 0.9
2.7 0.2 5.0 0.2 0.2 0.4 11.2 3.8 2.4 0.5 35.4 5.4 0.8 0.9
3.7 0.4 4.0 0'.2 0.3 0.4 11.3 1.6 1.7 0.5 24.5 4.0' 0.6 0.4 0.3 10:.1 21.3 2.6 ....
~.1 0.5. ~.5 0.2 0.2 0.1 13.3 2.1 1.6 0.2 26.6 3.8 0.5 0.4 0~6 6.8 17.8 3.7 1.7
2.8 0.3 5.3 0.3 0.2 0.1 18.0 2.2 1.5 0.3 30.5 4.3 0.6 0.3 0.5 5.3 20.2 4.2 t.1
9:2 19.8 1.5 ....
TABLE 2 Amounts of free glycerol at differen~ stages of hydro.lysis
Milk fat--200 mg
1VIono
(Moie%) 1.0 6.5 1.5 4.3
ciable q u a n t i t y during the course of the hydrolysis, the resulting monoglyeerides axe meaningless with respect to the original positions of the f a t t y acids. SchSnheyder and V o l q v a r t z (17) have shown that in t r i p r o p i o n a t e the f a t t y acid in the two position is not split by p a n creatic lipase and they did not observe any migration f r o m this position. BorgstrSm (3), using labelled f a t t y acids, showed that there is an interchange between free f a t t y acids and glyeerides during hydrolysis, but mostly in the one and three positions and very little, if any, in the two position. F i g u r e 2 shows the rates of hydrolysis of shnple and of mixed triglycerides.
F a t sample
2~ Hydro]ysls
Stage Digestion of hytime drolysis
Glyeerol
On in) 0 15 50
0 % 2~
(~gJ 0.0 2.2 10.2
0 8 27
0 % 2/a
0.0 0.6 3.4
i:9 17.0 3.5 1.1
W i t h both types of compounds the reactions proceed r a p i d l y to about 65% hydrolysis and much more slowly thereafter. O f course, those triglycerides which are not liquid at the digestion temperature, i.e., tripMmitin, are hydrolyzed very slowly. Since the evidence f o r the specificity of p a n c r e a t i c lipase f o r the external positions of glycerides is Convincing and is generally accepted, i f m i g r a t i o n f r o m the two position occurs at a r a t e equal to or f a s t e r than the rate of hydrolysis, hydrolysis should proceed at a u n i f o r m rate to completion. The f a c t that hydrolysis slows down substantially at about 65% is strong evidence that migration f r o m the two position is not occurring to an extent, during the early stages of the reaction, that would invalidate the technique as a means of studying glyceride structure. Application to a fat of known structure. Because the conditions of digestion p r o p o s e d f o r the structural analysis of milk f a t are somewhat different f r o m those used generally on other f a t s - - o n e - t h i r d hydrolysis instead of 6 0 % - - i t seemed desirable to determine whether data confirming the known structure of a simpler f a t could be obtained with this procedure. P i g body f a t was chosen because it has been studied by several investigators (11, 14, 16, 19) and has been shown to have a unique structure as compared with other fats. The p r e p a r a t i o n
288
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ED TG. ] b--TRIBUTYRIN
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I d-TRICAPRO~N
[ e--TRILAURIN
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20
40 60 TIME (MINUTES)
80
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I00
l~m. 2. Rates of pancrestic lipase hydrolysis of simple and of mixed trlglycerides. and analysis of the fat were described earlier. Table 3 shows the composition of the glycerides obtained from the hydro]ysis of pig fat. Examination of the values for the mono- and triglycerides shows the distribution of the fatty acids in the glyceride molecules. The concentration of palmitic and myristic acids in the two position, and of the unsaturated acids in the ternfinal positions, is in accord with the previously determined structure of this fat. Also, it is apparent that the stage of hydrolys i s - o n e - t h i r d or two-thirds--is not critical with fats containing only long-chain acids. DISCUSSIOI~
The charge (5) that pancreatic lipase hydrolysis cannot be used to study glyceride structure in glycerides containing short-chain fatty acids as, for example, in cow milk fat, because of the different rates at which the acids are hydrolyzed, cannot be dismissed lig'htly. I f this conclusion is correct, all of the work to date on
nfilk fat is meaningless and perhaps some of the conclusions on other fats are suspect, even though Entressangles et al. (5) state that acids of chain length C= and above are hydrolyzed at the same rate. As stated in the introduction, the 2-monoglyceride, if it is representative of the triglyceride from which it is formed, is the critical compound in this technique and it is our belief that when this is so the rate at which the acids in the one and three positions are hydrolyzed is immaterial. The 2-monoglyceride resulting from partial hydrolysis of a triglyceride should be representative of the original triglyceride under the following conditions: (a) that the acids hydrolyzed are restricted to the terminal positions, i.e., that the enzyme is specific for these positions, (b) that there has not been preferential hydrolysis of a particular triglyceride species, (c) that there has not been a substantial amount of complete hydrolysis, and (d) that there has not been appreciable migration of the fatty acids from the two position to the terminal positions during the reaction. The evidence for the specificity of pancreatic lipase for the tel~ninal groups is convincing and well accepted (2, 10, 15, 17). I t was not deemed necessalw to provide additional support for this point. Whether there has been preferential hydrolysis of a particular triglyceride species can be judged by comparing the composition of the residual triglycerides with the original triglycerides. I f the residual triglycerides resemble closely the original triglycerides, then it is a reasonable assumption that there has not been preferential hydrolysis. The agreement in composition between these groups in the nfilk fat at one-third hydrolysis and in the pig fat at both stages is excellent. Thus, from the stand-
TABLE 3 Composition of pig body fa~ and of residual g]ycerides after pancreatic lipase digestion Residual glyeerides Fatty acid Original chain length fat
hydrolysis Tri
Di
aN hydrolysis IVloi~o.
Tri
Di
Mono
(Mole %) 12 14 15 16 16:1 18 18 : 1 18:2
0.1 2.2 0.1 32.6 2.8 16.2 39.4 6.7
0.3 2.1 0.0 31.1 2.4 18.0 38.1 8.0
0.3 3.0 0.2 43.1 3.2 11.4 3:2.0 7.0
0.5 5.0 0.3 70.4 3.9 3.8 12.9 3.2
0.1 2.2 0.1 33.0 2.5 18.7 35.4 8.0
1.0 4.1 0.4 41.0 3.3 9.8 33.0 7.4
0.5 5.0 0.2 69.2 4.2 2.7 14.8 3.5
289
LIPASE HYDROLYSIS OF MILK FAT
point of preferential hydrolysis, the monoglyccrides of the cow milk fat at one-third hydrolysis (Table 1) and of the pig fat (Table 3) are valid entities for studying glyeeride structure. The free glycerol data in Table 2 show the extent of complete hydrolysis. I n the cow milk fat at one-third hydrolysis about 90% of the glycerol is present in esterified form. Ideally, one would wish for complete absence of free glycerol. However, even with the pig fat, which contains only long-chain fatty acids, this condition did not occur. I n this connection, Mattson and Lutton (11) state that there is some hydrolysis from the two position--less than 10%. Whether this degree of complete hyd~'olysis is sufficient to render the technique-pancreatic lipase hydrolysis--useless with milk fat is debatable. Obviously, the results with respect to complete hydrolysis do not permit as precise interpretation as one would wish. However, the authors believe that this anmunt of complete hydrolysis does not invalidate the method. The evidence concerning nfigration from the two to the one or three positions is presumptive. I n view of the specificity of the enzyme for the ternfinal fatty acids, the migration from the two position becomes important in this connection, if it proceeds at a rate approaching that of the hydrolysis reaction. If such a rate of migration occurred, the fatty acids would move out of the two position and would be hydrolyzed sufficiently fast so that the rate of hydrolysis should be relatively uniform to completion. The rate curves in Figure 2 show that this does not happen and that even with tributyrin the reaction slows materially at about 65% hydrolysis. This suggests that the rate of hydrolysis after the terminal acids are hydrolyzed depends upon the rate of migration from the two position and that this rate of migration, even with butyric acid, is slow as compared to the rate of hydrolysis of the terminal acids. It appears, then, that whether one accepts pancreatic lipase hydrolysis as a valid technique for studying the structure of cow milk fat depends upon how one regards the amount of complete hydrolysis obtained. I f one insists that any appearance of free glycerol destroys the reliability of the technique, then all studies using it on natural fats are open to suspicion, because free glycerol appeared in the hydrolysis of pig fat, even though the results agreed remarkably well with those reported previously. On the other hand, if one looks to the other criteria, particularly to the
absence of preferential hydrolysis, it would seem that the technique has merit, at least to establish general relationships, even though it does not permit as precise interpretation as might be desired. The data in Table 1 at one-third hydrolysis show that the majority of the fatty acids seem to be uniformly distributed within the triglycerides as shown by comparisons between the mono- and triglycerides. The major exceptions are C4 and C~, which are predominantly in the terminal positions, and C1~, which tends to concentrate in the two position. These results vary slightly from those of McCarthy et al. (12), where comparable values are available. Their data show a concentration of C1, in the two position but not C1¢, and also a tendency, not apparent here, for the unsaturated acids to be in the external positions. Additional studies oil specific glyeeride groups from milk fat now in progress should reveal more detailed information on glyceride structure. A~KNOWLEDGlYfENT
Miss T. Dairiki gave valuable assistance with the analytical work. REI~ERElqCES
(1) AST, It. J., AND VANDE~ WAn, R. J. The Structural Components of Milk Triglycerides. J. Am. Oil Chemists.' See., 38: 67. 1961. (2) BORGSTR:hM, BE~GT. On the Mechanism of the Hydrolysis of Glycerides by Pancreatic Lipase. Acta Chem. Scandia., 7:557. 1953. (3) BO~GSTahM, B]~NGT. On the Mechanism of Pancreatic Lipolysis. of Glyeerides. Biochim. et Biophys. Acta, 13:491. 1954. (4) C.ARROLL,K. K. Separation of Lipid Classes by Chromatography on Florisil. J. Lipid Research, 2: 135. 196'1. (5) ENTI~ESSAIqGL~S,B., PASI~I¢O,L., SAVAKY,P., SARDA~ L., AI~-D ])EISNUEILLE.~ P.
Influence
de lu Nature des Chaines sur ]g Vitess'e de leur Hydrolyse par Lipase pancrea$ique. Bull. soc. chim. biol., 43: 581. 1961. (6) GA~DER, G. W., ANn JmNSEN, R. G. Specificity of Milk Lipase Toward the Primary Ester Groups of Some Synthetic Triglycerides. J. Dairy Sci., 43: 1762. 1960. (7) HIR.SCH, J., AND AH~s~S, E. It. The Separation of Complex Lipide Mixtures by the Use of Silicic Acid Chronlatography. J. Biol. Chem., 23'3: 311. 1958. (8) KUMAR, S., PYNADATH, T. I., &ND LALKA, K. Location of Butyric Acid in Bovine Milk Triglycerides. Biochim. et Biophys. Acta, 42: 373. 1960. (9) LA~BFA~T~ M.,
AND NI~ISH, A.
C.
Rapid
Method for Estimation of Glycerol in Fer-
290
(10)
(]1)
(12)
(13)
(14)
E. L. JACK ET AL mentation Solutions. Canadian J. ICesearch, 28: 83. 1950. MATTS0N, F. It., AND B~CK, L. W. The Specificity of Pancreatic Lipase for the P r i m a r y Hydroxyl Groups of Glycerides. J. Biol. C.hem., 219: 735. 1956. MA~TS0N, F. H., AN]) Lt~TON, E. S. The Specific Distribution of F a t t y Acids in the Glycerides o{ Animal anal Vegetable Fats. J. Biol. Chem., 233: 868. 1958. MCCARTHY, R. D., PATTO~, STUART, AND EVANS, LAUtCA. Structure and Synthesis of Milk Fat. II. F a t t y Acid Distribution in the Triglycerides. J. Dairy Sci., 43: 1196. 1960. Q u m ~ N , P., AN]) W~Is]~a, H. J. Separation of Mono-, Di-, and Triglycerides ~ in Monoglyceride Concentrates. J. Am. Oil Chemists' Soc., 35: 326. 1958. R~ISE~, R., AI,rD ~AMAKRISHNA REDDY, H. G.
(15)
(16)
(17)
(18) (19)
The Glyceride Structure of Swine Depot Fat. J. Am. Oil Chemists' So c., 36: 97. 1959. S&VARY, P., AND D~SN-UELI.,E;, P. Sur quelques ~l~ments de Sp~cificite pendant 1 'Hydrolyse enzymatique des Triglycerldes. Biochim. et Biophys~ Acta, 21: 349. 1956. SAVARY, P., FSANZY, J., Awl) DKSNU~LL~,, P. Emplol de ]a Lipase pancreatique pour l ' E t u d e de ]a Structure des Corps naturelcs. Biochim. et Biophys. Acta, 24: 41¢. 1957. SC~SNHE~Z])Ba, F., AN]) VOL,QVA]~TZ, •. Studies on Lipolytic Enzyme Action. I I I . Hydrolysis o f Tripropionyl Glycerol. Biochim. et Biophys. Acta, 8: 407. 19'52. S~IT~, L. M. Quantitative F a t t y Acid Analysis of Milk F a t by Gas Liquid Chromatography. J. Dairy Sci., 44: 607. 1961. YOUNOS, C. G. Glyceride Structure of Fats. J. Am. Oil Chemists' Soc., 36: 664. 1959.
LIPASE HYDROLYSIS OF COW MILK FAT 1 C. P. FREEMAN,
L. M.
SMITH,
AN]) J. B. MICKLE
~
Department of Food Science and Technology, University of California, Davis SUMMARY
Knowledge of the position of individual fatty acids within the trig'lycerides is necessary to the understanding of the utilization of a fat. Pancreatic lipase hydrolysis to convert triglycerides to 2-monoglycerides has been used to study this type of glyceride structure in roany food fats, but it has recently been claimed that it cannot be used with cow milk fat because butyric acid is hydrolyzed more rapidly than the other acids. This study was undertaken to determine if there were conditions under which this technique could be applied validly to milk fat. The criteria set forth for the applicability of this technique were: (a) nonpreferential hydrolysis of triglyceride species; (b) absence of a substantial amount of complete hydrolysis; and (c) absence of a significant amount of acyl migration during the hydrolysis reaction. Although there was no evidence of preferential hydrolysis at the one-third stage, there was some complete hydrolysis, and acyl migration did not appear to be occurring at a significant rate. The validity of the procedures employed was demonstrated on a fat of known and unique structure, pig body fat. Therefore, the technique can give ~ s u l t s that may be used to establish general relationships. I n cow milk fat the majority of the fatty acids were found to be uniformly distributed within the glyceride, except for C, and C~, which are predominantly in the external positions, and C~, which tends to concentrate in the two position.
Knowledge of the position of the individual fatty acids within the triglycerides is important to the understanding of the synthesis, digestion and metabolism, and technology of fat. Recent developments in analytical techniques have made possible the acquisition of this knowledge for a nmnber of fats (1, 11, 12, 16, 19). The key technique in such studies is the application of controlled lipolysis to convert triglyeerides to monoglycerides. I t has been known for some time that pancreatic lipase hydrolyzes triglycerides to partial glycerides, but it remained for Mattson and Beck (10) and Savary et al. (15) to demonstrate that the action of the enzyme is primarily on the terminal fatty acid groups, and that the monoglycerides resulting are predominantly 2-monoglyeerides. Thus, by determining the fatty acids in the nmnoglyeerides, the position of the fatty acids in the triglyeerides is revealed. Other reports have confirmed the specificity of pancreatic and also nfilk lipases for the terminal acid groups (2, 6, 17). Received for publication December 17, 1962. Supported in part by PHS Research Grant A4250 from National Institute of Physiological Chemistry, Public Health Service. Postdoctoral fellow on sabbatical leave from Oklahoma State University, Stillwater, Oklahoma, 1961.
I n general, studies using this technique have shown that there is a nonrandom distribution of some of the fatty acids within the triglycerides, with the unsaturated fatty acids tending to be characteristically in the two position except for pig body fat, where they are in the one or three positions. Studies on cow milk fat using pancreatic lipase hydrolysis have not been sufficiently comprehensive for firm conclusions to be drawn. McCarthy et al. (12) reported results on milk fat produced from cows with normal feeding and after inanition, and related the composition to that of blood and body fat. They found that myristic acid (C1~) tends to concentrate in the two position and the C~ unsaturated acids in the external positions. Thus, from these data cow nfilk fat resembles lard in this respect. However, these investigators did not determine the lower fatty acids--C, and Co--and it is difficult to draw over-all conclusions. The same statement applies to the results by Ast and Vander Wal (1). Their conclusions are in agreement with those of McCarthy et al., but again the data for the lower fatty acids are questionable or lacking. A short paper by K u m a r et al. (8) deals with butyric acid (C,), which they find to be located ahnost exclusively in the one or three positions. Desnuelle's group, Entressangles et al. (5), has criticized this latter paper as the result of 284
LIPASE
HYDROLYSIS
their investigation of the effect of fatty acid chain length on the rate of hydrolysis by pancreatic lipase. They show that the short-chain acids are hydrolyzed nmre rapidly than the long-chain and that C~ is hydrolyzed the most rapidly of all. They state that it is thus impossible, as K u m a r et a l. (8) have claimed, to deternfine the position of the short-chain acids in the glyeerides of milk fat with a technique based on the positional specificity of lipase. Because this technique is being widely used in the study of glyceride structure, and its use is being extended to milk fat., where it offers a method of establishing the glyeeride structure of this complex fat, it becomes necessary to determine the validity of this statement. I t is a common observation, as documented by E'ntressangles et al. (5), that butyric acid is hydrolyzed more rapidly than the longerchain acids in both simple and nfixed glycerides, and that there is some difference in rates among fatty acids generally, depending upon chain length and other factors. However, since the monoglyceride formed is the critical compound in this analysis, the essential point is whether the monogIyceride is representative of the fat as a whole. I t is our belief that if the hydrolysis reaction is carried out in such a way that: (a) there is no preferential hydrolysis of any partitular triglyceride species, (b) a substantial amount of complete hydrolysis of the glycerides does not occur, and- (c) there is no appreciable acyl migration from the two position during the course of the reactions; then, under these conditions, the resulting monoglycerides are a reliable index of the positional coufigur3.tion of the fat. Experiments to test these criteria of the applicability of pancreatic lipase hydrolysis to cow milk fat are reported. EXPERIlVIENTAL PROCEDURE
I t seemed desirable to work initially with known materials in establishing whether the conditions set forth above could be attained. A nlixture of triglycerides containing butyric, caproic, caprylic, laurie, myristic, pahnitie, stearie, and oleic acids was chosen to simulate milk fat in range of fatty acid chain length and in amounts. Hydrolysis of this mixture as simple triglyeerides using pancreatic lipase was not satisfactory, because the long-chain group was not liquid at the temperature of hydrolysis, thus reducing greatly, or inhibiting entirely, its hydrolysis. Consequently, the nfixture was interesterified and satisfactory perforlnance was obtained. From prelinfinary
OF
MILK
FAT
285
trials with this interesterified glyceride mixture, conditions of hydrolysis were established which promised to be reliably applicable to inilk fat. Cow milk fat was then analyzed by the following procedures: After separating the triglyeerides from the crude fat by column chromatography, these were digested with pancreatie lipase to an appropriate stage as established by the preliminary trials, the digested mixture was separated into the free fatty acids and the different glyceride classes by colmnn chromatography, and the acids from the individual groups were then methylated and analyzed by GLC. To confirm the validity of the procedures, pig body fat, which has been thoroughly studied and which has been shown to have a characteristic positional patting, was similarly mlalyzed. Methods. Preparation of the fats. Cow milk fat was obtained by churning cream and melting and filtering the fat according to the procedure described by Smith (18). The pig body fat was taken from raw pork obtained locally. The back fat was homogenized 3-5 rain at room temperature in a Waring Blendor with 20 vol of CH~C1-MeOH (2:1) as solvent per gram of fat. The mixture was fltered through sintered glass and the solvent removed under reduced pressure. The dried lipid was re-extracted with 10 vol of CH~C1-MeOII solvent. The triglycerides from both the milk fat and the pig fat were separated from other materials as follows: Two grams of fat was taken up in a small volume o~ hexane and applied on to a colunto (140 g; 56 X 2.5 cm) of Florisil which had been oven-dried at 110 C for 24 hr. The column was eluted successively with 250 nfl of hexane, 1,500 ml of 50% ether in hexale, and 1,000 ml of 75% ether in hexane. The first 400 ml of eluate was discarded, and the next 1,000 nil, containing the triglycerides, was collected in bulk. Subsequent eluate was collected in 200-ml fractions and exanfined by thin-la~er chromatography to insure that the triglycerides were completely recovered from the column, and to check whether diglyceride was beginning to appear in the eluate. Those fractions containing triglycerides were added to the bulk, and the solvent removed at 0 C under reduced pressure. Pancreatic lipase digestion. The digestion nfixture contained 2.0 lnl of 45% CaCI~, 25 ml of 1.0 ~ NH~C1, and 10 ml of a 4% aqueous suspension of ground pork pancrease ~ which h3d been extracted previously with ethyl ether. 3 Mann Research Laboratories. Inc., New York
6, N . Y .
286
E.L. J~OK ~T ~L
Concentrated NH,OH was added to bring the p H to 8.0. This mixture was diluted to 100 ml with H~O, warmed to 40 C, and added to 0.1-0.3 g of triglyceride substrate. Digestion was carried out at 40 C with constant stirring and addition of NH~OI{ to maintain the p i t at 7.8-8.2. The reaction was stopped at an a p p r o p r i a t e stage by acidifying to p i t 2.0 with HC1. The glycerides and f a t t y acids were then extracted three times with 300 ml of ethyl ether-hexane (1:1). This extraction procedure recovered over 90% of the butyric acid and 100% of the other acids and glycerides. Separation of glyceride cl,asses. The silicic acid or silica gel eolunms, as described by Itirsch and Ahrens (7) or by Quinlin and Weiser (13), have been used most conmmnly to separate glyceride classes. W i t h milk fat, we were unable to get consistently satisfactorily distinct and quantitative separations with these procedures. However, the Florisil column technique of Carroll (4) was modified to give satisfactory results. The following procedures were adopted: Florisil was oven-dried at 110 C for 24 hr and 12 g was packed as a slurry in hexane in a colmnn (26 × 1 cm). The load was 100-150 mg. The eluting solvents were 250 ml of 60% ethyl ether in hexane, followed by 200 ml of 0.35% methanol in ethyl ether, then by 150 ml of 5.0% methanol in ethyl ether, and finally by 100 ml of 4% acetic acid in ethyl ether. These solvents remove tri-, di-, and monoglycerides, and f a t t y acids, in that order. Figure 1 shows the sharpness of separation of the different glyceride groups in a nfilk fat hydrolysate. Mcthylation and GLC. A f t e r the extracted lipids wer e separated into classes by the column chronmtographic procedure described above, the f a t t y acids front each glyceride class were methi
i
i
~ 6 o % ETHERIN HEXANE
,1~0.35~/o MeOHIN ETHER11--~5% MeOHtN ETHER~ I I
1 I I
F
D~-
TRY-
1 MONOGLYCERIDES
i I
I i
~0
I0
20
_
30 FRACTION
40
50
60
FrG. 1. Separation of glycerides i~t ~ milk fat hydrolysat~ on Florisil c o l u m n .
ylated and analyzed by GLC as described by Smith (18). RESULTS
Experiments with cow milk fat. Evidence bearing on perferential hydrolysis. In pancreatic lipase hydrolysis, the digestion is usually stopped when some fraction of the total f a t t y acids has been hydrolyzed. F r e quently, this is at about 60% hydrolysis. Among the residual materials are free fatty acids, mono-, di-, and triglyeerides. I f the enzyme has not preferentially attacked any pal~icular triglyeeride species, the residual triglycerides should resemble closely in composition the triglycerides from the original fat. PreliminaIT trials with the interesterified mixture showed this condition to obtain when 30-40% of the fatty acids had been hydrolyzed. Consequently, milk f a t was hydro]yzed and analyzed as described earlier. Digestions were carried to the one-third and two-thirds stages for comparison. Table 1 shows the results. The agreement between the composition of the triglycer{des of the original fat and that of the residual triglyeerides is remarkably close at one-third hydrolysis, indicating that at this stage there has not been significant preferential hydrolysis among the f a t t y acids at the terminal positions. A t the two-thirds stage the agreement is not quite as good, particularly with butyric acid. Rate of attainment of complete hydrolysis. A satisfactmT measure of complete hydrolysis is the appearance of free glycerol. Consequently, measurements of the amount of free glycerol were made by the method of Lambeth and Neish (9) at different stages in the digestion process. The prelimina~T trials with interesterified glycerides showed that only small amounts of free glycerol appeared up to 3040% hydrolysis. Accordingly, determinations of free glycerol were made at one-third and two-thirds hydrolysis. Table 2 shows the results obtained on both the milk fat and the pig fat. Assuming the average chain length of the f a t t y acids in milk fat to be approximately 14 carbon atoms, the amount of free glycerol produced at one-third hydrolysis is about 8% of the total glycerol. The molaz" amounts of free glycerol for pig fat at one-third hydrolysis are approximately 2-3% and 11-12% at two-thirds hydrolysis. E~idence bearing on acyl migration. Migration of f a t t y acids among the positions within glycerides is known to occur under certain conditions and if the acids in the two position migrate to the external positions in any appre-
LIPASE
HYDROLYSIS
TABLE
OF
MILK
FAT
287
1
Composition of cow milk fat and of residual glycerides after pancreatic lipase hydrolysis l~atty acid chain length 4 6 8 10 10 : 1 12 12:1 13 14 br. 14 14:1
15 16 br. 16 16:1 17 br.
Residual glyeerides OriginM fat 11.1 5.4 2.1 4.4 0.4 4.1 (}.2 0.1 0.1 11.7 1.5 1.3 0.1 23.7 2.6 0.6
17 18 br. 18
0.5 0.3 8.8
18:1
18.6
18:2 I8:3
1.6 0.8
Hydrolysis Tri
Di
Pig fat--200 mg
T1d
Di
Mono
5.9 4.1
1:6
9.7 5.2
9.3 5.1
2.0
1.o
1.0
1.3
1.4
0.6
4.5 0.6 4.1 0.3 0.3 0.1 11.8 1.8 1.1 0.1 24.4 2.9 0'N 0.2
4.6 0.5 4.~ 0.2 0.1 0.~ 14.0 2.0 1.5 0.5 2'4.4 3.6 0.3 0.1 0.2 6.9 17.3 2.2 0.9
2.7 0.2 5.0 0.2 0.2 0.4 11.2 3.8 2.4 0.5 35.4 5.4 0.8 0.9
3.7 0.4 4.0 0'.2 0.3 0.4 11.3 1.6 1.7 0.5 24.5 4.0' 0.6 0.4 0.3 10:.1 21.3 2.6 ....
~.1 0.5. ~.5 0.2 0.2 0.1 13.3 2.1 1.6 0.2 26.6 3.8 0.5 0.4 0~6 6.8 17.8 3.7 1.7
2.8 0.3 5.3 0.3 0.2 0.1 18.0 2.2 1.5 0.3 30.5 4.3 0.6 0.3 0.5 5.3 20.2 4.2 t.1
9:2 19.8 1.5 ....
TABLE 2 Amounts of free glycerol at differen~ stages of hydro.lysis
Milk fat--200 mg
1VIono
(Moie%) 1.0 6.5 1.5 4.3
ciable q u a n t i t y during the course of the hydrolysis, the resulting monoglyeerides axe meaningless with respect to the original positions of the f a t t y acids. SchSnheyder and V o l q v a r t z (17) have shown that in t r i p r o p i o n a t e the f a t t y acid in the two position is not split by p a n creatic lipase and they did not observe any migration f r o m this position. BorgstrSm (3), using labelled f a t t y acids, showed that there is an interchange between free f a t t y acids and glyeerides during hydrolysis, but mostly in the one and three positions and very little, if any, in the two position. F i g u r e 2 shows the rates of hydrolysis of shnple and of mixed triglycerides.
F a t sample
2~ Hydro]ysls
Stage Digestion of hytime drolysis
Glyeerol
On in) 0 15 50
0 % 2~
(~gJ 0.0 2.2 10.2
0 8 27
0 % 2/a
0.0 0.6 3.4
i:9 17.0 3.5 1.1
W i t h both types of compounds the reactions proceed r a p i d l y to about 65% hydrolysis and much more slowly thereafter. O f course, those triglycerides which are not liquid at the digestion temperature, i.e., tripMmitin, are hydrolyzed very slowly. Since the evidence f o r the specificity of p a n c r e a t i c lipase f o r the external positions of glycerides is Convincing and is generally accepted, i f m i g r a t i o n f r o m the two position occurs at a r a t e equal to or f a s t e r than the rate of hydrolysis, hydrolysis should proceed at a u n i f o r m rate to completion. The f a c t that hydrolysis slows down substantially at about 65% is strong evidence that migration f r o m the two position is not occurring to an extent, during the early stages of the reaction, that would invalidate the technique as a means of studying glyceride structure. Application to a fat of known structure. Because the conditions of digestion p r o p o s e d f o r the structural analysis of milk f a t are somewhat different f r o m those used generally on other f a t s - - o n e - t h i r d hydrolysis instead of 6 0 % - - i t seemed desirable to determine whether data confirming the known structure of a simpler f a t could be obtained with this procedure. P i g body f a t was chosen because it has been studied by several investigators (11, 14, 16, 19) and has been shown to have a unique structure as compared with other fats. The p r e p a r a t i o n
288
E
I
I
I
I
I
I
I
I
L. J A C K E T A L
I
I
80
>-~
//
o ac
//
40
ED TG. ] b--TRIBUTYRIN
~" ~ /
I c--TmOLEm
/ /// / ~"
/f
//
>-:z
,,/. / / e
I d-TRICAPRO~N
[ e--TRILAURIN
//// O~
20
40 60 TIME (MINUTES)
80
I
I
I00
l~m. 2. Rates of pancrestic lipase hydrolysis of simple and of mixed trlglycerides. and analysis of the fat were described earlier. Table 3 shows the composition of the glycerides obtained from the hydro]ysis of pig fat. Examination of the values for the mono- and triglycerides shows the distribution of the fatty acids in the glyceride molecules. The concentration of palmitic and myristic acids in the two position, and of the unsaturated acids in the ternfinal positions, is in accord with the previously determined structure of this fat. Also, it is apparent that the stage of hydrolys i s - o n e - t h i r d or two-thirds--is not critical with fats containing only long-chain acids. DISCUSSIOI~
The charge (5) that pancreatic lipase hydrolysis cannot be used to study glyceride structure in glycerides containing short-chain fatty acids as, for example, in cow milk fat, because of the different rates at which the acids are hydrolyzed, cannot be dismissed lig'htly. I f this conclusion is correct, all of the work to date on
nfilk fat is meaningless and perhaps some of the conclusions on other fats are suspect, even though Entressangles et al. (5) state that acids of chain length C= and above are hydrolyzed at the same rate. As stated in the introduction, the 2-monoglyceride, if it is representative of the triglyceride from which it is formed, is the critical compound in this technique and it is our belief that when this is so the rate at which the acids in the one and three positions are hydrolyzed is immaterial. The 2-monoglyceride resulting from partial hydrolysis of a triglyceride should be representative of the original triglyceride under the following conditions: (a) that the acids hydrolyzed are restricted to the terminal positions, i.e., that the enzyme is specific for these positions, (b) that there has not been preferential hydrolysis of a particular triglyceride species, (c) that there has not been a substantial amount of complete hydrolysis, and (d) that there has not been appreciable migration of the fatty acids from the two position to the terminal positions during the reaction. The evidence for the specificity of pancreatic lipase for the tel~ninal groups is convincing and well accepted (2, 10, 15, 17). I t was not deemed necessalw to provide additional support for this point. Whether there has been preferential hydrolysis of a particular triglyceride species can be judged by comparing the composition of the residual triglycerides with the original triglycerides. I f the residual triglycerides resemble closely the original triglycerides, then it is a reasonable assumption that there has not been preferential hydrolysis. The agreement in composition between these groups in the nfilk fat at one-third hydrolysis and in the pig fat at both stages is excellent. Thus, from the stand-
TABLE 3 Composition of pig body fa~ and of residual g]ycerides after pancreatic lipase digestion Residual glyeerides Fatty acid Original chain length fat
hydrolysis Tri
Di
aN hydrolysis IVloi~o.
Tri
Di
Mono
(Mole %) 12 14 15 16 16:1 18 18 : 1 18:2
0.1 2.2 0.1 32.6 2.8 16.2 39.4 6.7
0.3 2.1 0.0 31.1 2.4 18.0 38.1 8.0
0.3 3.0 0.2 43.1 3.2 11.4 3:2.0 7.0
0.5 5.0 0.3 70.4 3.9 3.8 12.9 3.2
0.1 2.2 0.1 33.0 2.5 18.7 35.4 8.0
1.0 4.1 0.4 41.0 3.3 9.8 33.0 7.4
0.5 5.0 0.2 69.2 4.2 2.7 14.8 3.5
289
LIPASE HYDROLYSIS OF MILK FAT
point of preferential hydrolysis, the monoglyccrides of the cow milk fat at one-third hydrolysis (Table 1) and of the pig fat (Table 3) are valid entities for studying glyeeride structure. The free glycerol data in Table 2 show the extent of complete hydrolysis. I n the cow milk fat at one-third hydrolysis about 90% of the glycerol is present in esterified form. Ideally, one would wish for complete absence of free glycerol. However, even with the pig fat, which contains only long-chain fatty acids, this condition did not occur. I n this connection, Mattson and Lutton (11) state that there is some hydrolysis from the two position--less than 10%. Whether this degree of complete hyd~'olysis is sufficient to render the technique-pancreatic lipase hydrolysis--useless with milk fat is debatable. Obviously, the results with respect to complete hydrolysis do not permit as precise interpretation as one would wish. However, the authors believe that this anmunt of complete hydrolysis does not invalidate the method. The evidence concerning nfigration from the two to the one or three positions is presumptive. I n view of the specificity of the enzyme for the ternfinal fatty acids, the migration from the two position becomes important in this connection, if it proceeds at a rate approaching that of the hydrolysis reaction. If such a rate of migration occurred, the fatty acids would move out of the two position and would be hydrolyzed sufficiently fast so that the rate of hydrolysis should be relatively uniform to completion. The rate curves in Figure 2 show that this does not happen and that even with tributyrin the reaction slows materially at about 65% hydrolysis. This suggests that the rate of hydrolysis after the terminal acids are hydrolyzed depends upon the rate of migration from the two position and that this rate of migration, even with butyric acid, is slow as compared to the rate of hydrolysis of the terminal acids. It appears, then, that whether one accepts pancreatic lipase hydrolysis as a valid technique for studying the structure of cow milk fat depends upon how one regards the amount of complete hydrolysis obtained. I f one insists that any appearance of free glycerol destroys the reliability of the technique, then all studies using it on natural fats are open to suspicion, because free glycerol appeared in the hydrolysis of pig fat, even though the results agreed remarkably well with those reported previously. On the other hand, if one looks to the other criteria, particularly to the
absence of preferential hydrolysis, it would seem that the technique has merit, at least to establish general relationships, even though it does not permit as precise interpretation as might be desired. The data in Table 1 at one-third hydrolysis show that the majority of the fatty acids seem to be uniformly distributed within the triglycerides as shown by comparisons between the mono- and triglycerides. The major exceptions are C4 and C~, which are predominantly in the terminal positions, and C1~, which tends to concentrate in the two position. These results vary slightly from those of McCarthy et al. (12), where comparable values are available. Their data show a concentration of C1, in the two position but not C1¢, and also a tendency, not apparent here, for the unsaturated acids to be in the external positions. Additional studies oil specific glyeeride groups from milk fat now in progress should reveal more detailed information on glyceride structure. A~KNOWLEDGlYfENT
Miss T. Dairiki gave valuable assistance with the analytical work. REI~ERElqCES
(1) AST, It. J., AND VANDE~ WAn, R. J. The Structural Components of Milk Triglycerides. J. Am. Oil Chemists.' See., 38: 67. 1961. (2) BORGSTR:hM, BE~GT. On the Mechanism of the Hydrolysis of Glycerides by Pancreatic Lipase. Acta Chem. Scandia., 7:557. 1953. (3) BO~GSTahM, B]~NGT. On the Mechanism of Pancreatic Lipolysis. of Glyeerides. Biochim. et Biophys. Acta, 13:491. 1954. (4) C.ARROLL,K. K. Separation of Lipid Classes by Chromatography on Florisil. J. Lipid Research, 2: 135. 196'1. (5) ENTI~ESSAIqGL~S,B., PASI~I¢O,L., SAVAKY,P., SARDA~ L., AI~-D ])EISNUEILLE.~ P.
Influence
de lu Nature des Chaines sur ]g Vitess'e de leur Hydrolyse par Lipase pancrea$ique. Bull. soc. chim. biol., 43: 581. 1961. (6) GA~DER, G. W., ANn JmNSEN, R. G. Specificity of Milk Lipase Toward the Primary Ester Groups of Some Synthetic Triglycerides. J. Dairy Sci., 43: 1762. 1960. (7) HIR.SCH, J., AND AH~s~S, E. It. The Separation of Complex Lipide Mixtures by the Use of Silicic Acid Chronlatography. J. Biol. Chem., 23'3: 311. 1958. (8) KUMAR, S., PYNADATH, T. I., &ND LALKA, K. Location of Butyric Acid in Bovine Milk Triglycerides. Biochim. et Biophys. Acta, 42: 373. 1960. (9) LA~BFA~T~ M.,
AND NI~ISH, A.
C.
Rapid
Method for Estimation of Glycerol in Fer-
290
(10)
(]1)
(12)
(13)
(14)
E. L. JACK ET AL mentation Solutions. Canadian J. ICesearch, 28: 83. 1950. MATTS0N, F. It., AND B~CK, L. W. The Specificity of Pancreatic Lipase for the P r i m a r y Hydroxyl Groups of Glycerides. J. Biol. C.hem., 219: 735. 1956. MA~TS0N, F. H., AN]) Lt~TON, E. S. The Specific Distribution of F a t t y Acids in the Glycerides o{ Animal anal Vegetable Fats. J. Biol. Chem., 233: 868. 1958. MCCARTHY, R. D., PATTO~, STUART, AND EVANS, LAUtCA. Structure and Synthesis of Milk Fat. II. F a t t y Acid Distribution in the Triglycerides. J. Dairy Sci., 43: 1196. 1960. Q u m ~ N , P., AN]) W~Is]~a, H. J. Separation of Mono-, Di-, and Triglycerides ~ in Monoglyceride Concentrates. J. Am. Oil Chemists' Soc., 35: 326. 1958. R~ISE~, R., AI,rD ~AMAKRISHNA REDDY, H. G.
(15)
(16)
(17)
(18) (19)
The Glyceride Structure of Swine Depot Fat. J. Am. Oil Chemists' So c., 36: 97. 1959. S&VARY, P., AND D~SN-UELI.,E;, P. Sur quelques ~l~ments de Sp~cificite pendant 1 'Hydrolyse enzymatique des Triglycerldes. Biochim. et Biophys~ Acta, 21: 349. 1956. SAVARY, P., FSANZY, J., Awl) DKSNU~LL~,, P. Emplol de ]a Lipase pancreatique pour l ' E t u d e de ]a Structure des Corps naturelcs. Biochim. et Biophys. Acta, 24: 41¢. 1957. SC~SNHE~Z])Ba, F., AN]) VOL,QVA]~TZ, •. Studies on Lipolytic Enzyme Action. I I I . Hydrolysis o f Tripropionyl Glycerol. Biochim. et Biophys. Acta, 8: 407. 19'52. S~IT~, L. M. Quantitative F a t t y Acid Analysis of Milk F a t by Gas Liquid Chromatography. J. Dairy Sci., 44: 607. 1961. YOUNOS, C. G. Glyceride Structure of Fats. J. Am. Oil Chemists' Soc., 36: 664. 1959.