THE DIGESTION IN VITRO OF TRIGLYCERIDES BY PANCREATIC LIPASE

BY F. H. MATTSON AND L. W. BECK

(From the Research Division, The Procter and Gamble Company, Cincinnati, Ohio)

(Received for publication, September 22, 1954)

The formation of partial glycerides by the hydrolysis in vitro of triglyc- erides with pancreatic lipase was first reported by Artom and Reale (1) and then again several years later by Frazer and Sammons (2). In these studies apparently no attention was paid to the isomeric form of the diges- tion products. Desnuelle et al. (3) further studied the hydrolysis of tri- glycerides and showed it to be a series of stepwise reactions from triglyceride to diglyceride to monoglyceride to glycerol, fatty acid being released at each step. The quantitative aspects of this work were weakened by their supposition that the hydrolysis was a random one. The pertinent liter- ature in this field has been the subject of an excellent review by Desnuelle (4).

The hydrolysis in vivo of triglycerides was reported by this laboratory (5) to be a directed one from triglyceride to 1,2-diglyceride to 2-monoglyceride. Partial conversion of 2-monoglyceride to the 1 isomer also appeared to occur in the intestine. It was likewise reported that at the conclusion of hydrolysis in vitro only the 1 isomer of monoglyceride was present. Sub- sequently, Borgstriim (6), using methods never previously applied to these types of materials, reported that there is selective formation of 1,2-diglyc- erides and 2-monoglycerides when triglycerides are hydrolyzed in vitro with pancreatic lipase.

In the studies reported here the course of hydrolysis in vitro was found to be the same as hydrolysis in vivo. From some of the characteristics of the reactions, it appears that lipase may be specific for the hydrolytic and esterification reactions occurring at the primary hydroxyl group positions of glycerol.

Methods

The digestion conditions used in these studies were essentially those de- scribed by Desnuelle et al. (3), except t,hat the buffer concentration was in- creased to 1.0 M. Unless otherwise indicated, the digestion mixture con-

sisted of 13.G ml. of 1.0 M ammonium chloride-ammonium hydroxide buffer adjusted to pH 8.0, 0.3 ml. of a 0.15 per cent aqueous solution of bile salts (Difco), 1.0 ml. of a 45 per cent aqueous calcium chloride solution, 2.3 ml. of an aqueous suspension of 156 mg. of steapsin (Difco), and 2.0 gm. of

115

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116 DIGESTION OF TRIGLYCERIDES

triglyceride. In all of the studies but one, “winterized” cottonseed oil* was the substrate used. During the digestion period (15 minutes) at 40” f 0.5”, the mixture was continuously agitated and a pH of 8.0 was main- tained by the addition of 4 M ammonium hydroxide.

In the studies on the effect of electrolyte concentration, the buffer was omitted, but otherwise the standard digestion procedure given above was used. Although the digests initially contained known concentrations of electrolyte, the addition of ammonium hydroxide to maintain the pH neces- sarily resulted in a dilution of the electrolyte. The more rapid the rate of hydrolysis, the greater the quantity of ammonium hydroxide that was needed, and hence the greater was the dilution of the electrolyte. To allow for this dilution, the amount of added ammonium hydroxide was measured and the final molar concentration of the electrolyte was calculated. From the initial and final molarity, the “average” molarity was calculated and this latter figure was used as a basis for comparison.

At the end of the digestion period the pH was adjusted to 2 with N HCl, 25 ml. of ethyl alcohol were added, and the lipides were extracted with ethyl ether. The ethyl ether solution was washed with water and the lipides were treated in the same manner as in the studies in vivo (5). The extracted aqueous layer, to which were added the water washings of the ethyl ether solutions, was retained for glycerol analysis.

The free fatty acid content of the lipides was determined by titration. l- and 2-monoglycerides were determined by the isomerization and analyti- cal procedures described by Martin (7). Free glycerol was determined by the method of Lambert and Neish (8). Microadaptations of the neutrali- zation equivalent technique, the Wijs iodine value procedure, and the hy- droxyl value method of the American Oil Chemists’ Society were used (9).

In certain of the studies fractionation of the lipides was carried out. The free fatty acids were isolated first with Amberlite IRA-400, according to the procedure described by Cason et al. (10). The method of “completion of squares” (ll), by solvent extraction (multistage separatory funnel separa- tion), was employed for the isolation of monoglycerides and diglycerides. For this purpose, the lipides of the digest, from which the free fatty acids had been removed by Amberlite treatment, were passed through a 4 X 4 “completion of squares” process with 80 per cent ethanol-Skellysolve F as the solvent pair. Under these conditions the monoglycerides will be found in alcohol Fractions 3 and 4. The lipides in the Skellysolve Fraction 1, which are essentially all of the diglycerides and triglycerides, were then distributed in a 10 X 10 “completion of squares” process with 95 per cent

1 Cottonseed oil from which the higher melting glycerides have been removed by cooling and filtration of the liquid from the solid portions.

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F. H. MAlTSON AND L. W. BECK 117

mebhanol and isooctane as the solvent pair. In this system the diglycerides will be found chiefly in alcohol Fractions 4,5,6, and 7.2

Results

Type of Mono- and Diglyceride Isomers Formed

In the portion of the studies carried out in vitro that was referred to in the earlier report (5), digestion periods of 60 minutes or more were used. Subsequently, digests carried out for shorter periods of time have been

TABLE I

Proportion of Isomers of Monoglyceride Found &fter Various Periods of Hydrolysis of Trig1 ycerides

Length of digestion 1-Monoglyceride 2.Monoglyceride Total monoglyceride Per cent as 2-monoglyceride

min. wcighl per cent weight ger cm! wcighf ficr cd

15 8.1 8.7 16.8 30 15.7 10.1 25.8 60 28.1 3.4 31.5

52 39 11

--

TABLE II

Digestion Products of B-Oleoyl Dipalmitin

Weight per cent

Free fatty acid. . . . . . . 34 Monoglyceride*................................... 17 Diglyceride....................................... 33 Triglyceride....................................... 16

* Consisting of 25 per cent 1-monoglyceride and 75 per cent 2-monoglyceride.

analyzed for l- and 2-monoglycerides. The results of one such series are given in Table I. Of the monoglycerides found at the end of 15, 30, and 60 minute digestion periods, 52, 39, and 11 per cent, respectively, were the 2 isomer. The decrease in the relative proportions of 2-monoglycerides with time is probably due to isomerization rather than to initial formation of l-monoglycerides, as indicated below in studies with 2-oleoyl dipalmitin as a substrate.

In order to check further on the formation of 2-monoglycerides, and to investigate the type of diglycerides formed, digestion of 30 minutes dura-

2 The authors wish to acknowledge the assistance of C. B. Stewart, of these lab- oratories, who developed these particular solvent separation methods.

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118 DIGESTION OF TRIGLYCERIDES

tion was carried out with 2-oleoyl dipalmitin as the substrate. The com- position of the lipides at the end of the digestion is given in Table II.

From this lipide mixture the free fatty acids, monoglycerides, and di- glycerides were isolated. Since the initial substrate had a known glyceride composition, it was possible to calculate what fatty acids, monoglycerides, and diglycerides could be formed by the various theoretical routes of split- ting. The values for the possible products and those found for the fractions isolated are given in Table III. From these data it can be seen that es-

TABLE III

Charucteristics oj Observed and Theoretical Digestion Products of 2-Oleoyl Dipalntitin

Purity Iodine value Mol. wt.

Free fatty acid fraction

Observed ......................... Palmitic* ......................... Oleic* .............................

per cent

100 8.7 263 0 256

90 282

Monoglyceride fraction

Observed ......................... iVonoolein* ....................... hIonopalmitin*. ...................

Diglgccride fraction

Observed, ........................ Oleoyl palmitin*. ................. Dipalmitin*. ......................

* Theoretical values.

sentially all of the free fatty acids were palmitic acid, the monoglycerides were monoolein, and the diglycerides were oleoyl palmitin. Thus, the course of hydrolysis is apparently 2-oleoyl diplamitin + 2-oleoyl-l- monopalmitin -+ 2-monoolein, palmitic acid being released at each step. Since the monoglyceride fraction consisted essentially of monoolein, it would appear that the I-monoglyceride present in the sample arose from the isomerization of %-monoolein to l-monoolein.

Factors Influencing Course of Digestion

The effect of varying the constjituents of the digest was investigated next. For ease of analysis in these studies the amounts of monoglyceride and of free fatty acid formed were used as criteria of the rate of hydrolysis.

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F. II. MAlTSON .4ND L. W. BECK 119

Electrolyte Concentration-In the first series of studies the effect of t,he electrolyte concentration on the rate of digestion was considered. The amounts of monoglyceride found after 15 minutes of hydrolysis, when the digest contained various concentrations of ammonium chloride, ammonium sulfat.c, sodium chloride, or glucose, arc shown in Fig. 1. In t hc abscncc of electrolyte the digestion proceeded quite slowly. As the concentration of electrolyte was increased, the rate of digestion passed through a maximum, then fell off with further increasing concentration. On the other hand, the addition of a non-electrolyte, glucose, had no effect on the rate of hy- drolysis. Although not shown, the formation of free fatty acid followed a similar pattern.

2.0 2.5 3.0

‘AVERAGE’ MOLARITY Fro. 1. Effect of electrolyte snd non-electrolyte concentrations on the enzymntic

formation of monoglycerides (MG) from triglycerides. X, NH&l; 0, N&l; A, (NH&SO1; 0, glucose.

Bile Salt Concentration-The effect cf varying the concentration of bile salts on the rate of digestion, as measured by the amount of monoglyceride and free fatty acid formed, is shown in Fig. 2. When 10 mg. of bile salts (0.05 per cent concentration) were added, the maximal rate of activity was obtained. At higher or lower concentrations the rate of hydrolysis was slower.

Enzyme Concentration-The effect of varying the amount of enzyme in the digest on the rate of monoglyceride and free fatty acid formation is shown in Fig. 3. As the amount of added enzyme increased, so did the amount of digestion products formed. However, the response was not linear but in the form of S-shaped curves.

Kinetics of Reaction

In the final series of studies, digestion periods of 5, 10, 15, 20, and 30 minutes were employed. The concentrations in moles per cent of the var- ious glyceride types at the end of these periods are shown in Fig. 4. At the end of 30 minutes of hydrolysis no free glycerol was present.

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120 DIGESTION OF TRIGLYCERIDES

The glyceride curves are the type that would be expected for a series of consecutive reactions. A plot of the length of time of hydrolysis against

0' IO 20 30 40 50 60

MlLLlGFjAMS OF BILE SALTS

Fra. 2. Effect of bile salt concentration on the enzymatic formation of mono- glycerides (MG) and free fatty acids (FFA) from triglycerides.

60- FFA

MG I

70

MILLIGRAMS OF ENZYME

FIG. 3. Effect of enzyme concentration on the enzymatic formation of mono- glycerides (MG) and free fatty acids (FFA) from triglycerides.

the log of moles of unhydrolyzed triglyceride gives a straight line, indicating the hydrolysis of triglyceride to diglyceride to be a pseudomonomolecular reaction. The rate constant was calculated to be 0.07 min.-‘.

Since the amount of monoglyceride formed is equal to the amount of diglyceride hydrolyzed, and as there was no appreciable monoglyceride hydrolysis, the monoglyceride values can be used in determining the rate

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F. H. MATTSON AND L. W. BECK 121

of diglyceride hydrolysis. During the initial period of digestion, 0 to 5 minutes, the rate of formation of monoglyceride, and hence of diglyceride hydrolysis, was 1.2 moles per cent per minute. This rate increased to 2 moles per cent per minute during the second 5 minutes of hydrolysis and remained relatively constant at this rate for the remainder of the time in- tervals studied. This constancy was obtained in spite of wide variations in the concentration of diglyceride during the course of the hydrolysis. Thus, the conversion of diglyceride to monoglyceride is indicated to be a zero order reaction having a velocity constant of approximately 2 moles per cent per minute.

MINUTES

Fro. 4. The course of the enzymatic hydrolysis of triglycerides. MG = mono- glyceride, DG = diglyceride, and I’G = triglyceride.

Attempts thus far made to determine the velocity constant for the reac- tion monoglyceride to glycerol have been unsuccessful. At the end of a 90 minute hydrolysis period, with triglyceride as the original substrate, the concent,ration of monoglyceride is still increasing and only 10 per cent of the glycerol present has been hydrolyzed to free glycerol. Apparently under these conditions the last step in the hydrolysis proceeds at a very slow rate.

DISCUSSION

The reason for the failure to recognize the formation of 2-monoglycerides in our earlier experiments was the use of hydrolysis periods of 60 minutes and longer. When shorter digestion periods were employed, as in these studies, considerable quantities of the 2 isomer were found. Although the monoglyceride initially formed is the 2 isomer, with longer exposure to these digestion conditions there is a proportionate increase in isomerization of 2-monoglyceride to 1-monoglyceride. The value of 11 per cent for the

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122 DIGESTION OF TRTGLPCERIDIXS

amount of the 2 isomer present after 60 minutes of hydrolysis is approxi- mately that for an equilibrium mixture of l- and 2-monoglycerides (7).

The resuhs of the studies both in vitro and in vivo still do not make it clear whether t,he conversion of 2-monoglyceride to t.he 1 isomer is solely enzymatic or merely the result of physical :t11d chcmic*al caonditions prcscnt during digestion. In the process of isolating the lipides isomerization could also occur, even though all efforts were made to minimize this possibility. Thus, the ratio of 1-monoglyceride to 2-monoglyceride at the completion of the hydrolysis period remains uncert,ain.

The results obtained when 2-oleoyl dipalmitin was used as a substrate further demonstrate that the hydrolysis of triglyceride is a directed one from triglyceride to 1,2-diglyceride to 2-monoglyceride. The figures ob- tained were not in absolute agreement with theoretical values, but these minor differences can be attributed to failure to isolate entirely pure frac- tions and to the possibility that esterification is taking place at the same time as hydrolysis. BorgstrGm (12) has shown that esterification does occur both in vitro and in vivo. However, the presence of calcium ions in the experiments reported here should keep any synthesis involving free fatty acids to a minimum, since these acids were held in an insoluble state as the calcium soaps.

A similar course of digestion for glycerides of short chain fatty acids has bx~~ reported by Schonheyder and Volqvartz (13). Using simple mono- glycerides, diglycerides, or triglycerides of propionic acid as substrates, they concluded that the hydrolysis of tripropionin was selectively to 1,2- dipropionin and then to 2-monopropionin which in turn was hydrolyzed very slowly. This route of hydrolysis was observed with both pancreatic lipase and liver esterase.

In the experiments reported here, in which the glycerol was esterified with long chain fatty acids, a selective formation of 1,2-diglycerides and 2-monoglycerides was also found. Similar results with respect to 2-mono- glyceride formation have been obtained in vivo with triglycerides consist- ing of short (14) and long (5) chain fatty acids. These results indicate that pancreatic lipase is specific for the esterified primary hydroxyl groups of glycerol.

If the enzyme is specific for the primary hydroxyl groups, any l-mono- glyceride that is present should also be hydrolyzed. Thus, there is some indication that little isomerization of 2-monoglycerides takes place under the digestion conditions. On the other hand, the considerably different physical properties of the monoglycerides, e.g. solubility, interfacial orien- tation, etc., as compared to those of diglycerides and triglycerides, could account for the extremely slow rate of hydrolysis of the monoglyceride.

Consideration of the reactions triglyceride to diglyceride and diglyceride

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F. II. MATTSON AND L. W. BECK 123

to monoglyceride would lead one to expect that both would be pseudomono- molecular reactions. The experimental values showed this to be true for the hydrolysis of triglycerides. However, the conversion of diglyceride to monoglyceride was found to be a zero order reaction during all but the ini- tial period of hydrolysis. During a portion of the first 5 minutes of hy- drolysis the reaction may have been first order. As the reaction proceeded, some other factor became rate-limiting and hence the change in order of reaction.

The change in reaction order of the diglyceride to monoglyceride reaction also would be expected to occur for the reaction triglyceride to diglyceride, if both were catalyzed by the same enzyme. However, the rate of tri- glyceride hydrolysis did not change over the course of the digestion. Thus, the possibility is indicated that two separate enzymes may be involved in the hydrolysis of triglycerides and diglycerides.

The unequivocal establishment of the kinetics of the hydrolysis of various isomers of diglycerides and monoglycerides and the effect of the surface activity of monoglycerides on hydrolysis can be determined only by using pure 1,2- and 1,3-diglycerides and l- and 2-monoglycerides as substrates. Such studies are currently being carried out in.our laboratory.

In most of these studies, t.he initial buffer concentration of 1 M was based on the results of preliminary experiments, indicating that such a high con- centration of buffer gave a fast rate of reaction. The experiments summa- rized in Fig. 1 demonstrate that this effect was a function of the electrolyte concentration and did not depend on the buffering action of the ammonium chloride. The fastest rate of hydrolysis was obtained with an initial elec- trolyte concentration of 2 M, which under the conditions described would give a final “average” molarity of 1.4. These observations may be a re- flection of the effect of the electrolyte concentration on the physical prop- erties of the enzyme, such as solubility or the extent of unfolding of the protein molecule with resulting changes in available reaction sites.

The results obtained when various concentrations of bile salts were used present two interesting points: The first is that even in the absence of bile salts hydrolysis proceeds quit’e rapidly and the rate is only slightly in- creased by their presence. The second is that hydrolysis is inhibited by higher concentrations of bile salts.

Under the conditions of these experiments bile salts appear to have a minor r81a in tdlc tligrst.ion of glycerides. In this connection it is interest- ing t,o note the wide variety of effects obtained by Wills (15) in his studies on the effect of various detergents and bile salts on the rate of hydrolysis by pancreatic lipase. Borgstrijm (16) has suggested that one function of the bile salts in vivo may he to lower the pH optimum of lipase so that it will more closely correspond with that in the intestinal tract.

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124 DIGESTION OF TRIGLYCERIDES

It will be noted that when the concentration of enzyme was varied the initial portion of the curve was not linear as would normally be expected. However, it should be recognized that the hydrolysis is not a single reaction but a series of consecutive reactions. In these experiments the amounts of monoglyceride and free fatty acid formed were used as the criteria of the rate of hydrolysis. The initial lag in these curves can therefore be attrib- uted to the reaction at low enzyme concentration being predominantly triglyceride to diglyceride. When viewed in this light, these are the type of curves that should be obtained under these conditions.

SUMMARY

The nature of the products and some of the factors influencing the hy- drolysis of triglycerides by pancreatic lipase in vitro have been studied. It was found that (a) hydrolysis was favored by a high electrolyte concentra- tion (1 to 2 M), (b) bile salts at low concentration caused a slight increase in the rate of hydrolysis but at higher levels were inhibitory, (c) increasing the amount of enzyme resulted in the formation of more digestion products, but the effect was not linear.

Digests of less than 60 minutes duration contained appreciable quantities of %monoglyceride, as well as 1-monoglyceride. If hydrolysis was allowed to proceed for 60 minutes, only the equilibrium mixture of l- and 2-mono- glyceride was present.

When 2-oleoyl dipalmitin was used as a substrate, the diglyceride formed was oleoyl palmitin and the monoglyceride was monoolein. Thus, the course of hydrolysis of triglycerides in vitro appears to be the same as that in vivo, namely, a series of directed stepwise reactions from triglyceride to 1,2-diglyceride to 2-monoglyceride. The 1-monoglyceride is the result of isomerization of the 2-monoglyceride.

The hydrolysis of triglyceride to diglyceride was found to be a first order reaction with a velocity constant of 0.07 min.-‘. The conversion of di- glyceride to monoglyceride was chiefly a zero order reaction with a velocity constant of 2 moles per cent per minute. The hydrolysis of monoglyceride to glycerol proceeded at such a slow rate that it was impossible to charac- terize this reaction.

It is suggested that the enzyme, lipase, is specific for esterified primary hydroxyl groups.

BIBLIOGRAPIIY

1. .4rtom, C., and Reale, L., Boll. Sot. ilal. biol. sper., 10, 883 (1935). 2. Frazer, A. C., and Sammons, H. G., Biochem. J., 39, 122 (1945). 3. Desnuelle, P., Naudet, RI., and Rouyier, J., Biochim. et biophys. a&, 2. 561

(1948). 4. Desnuelle, P., Bull. Sot. chim. biol., 33, 909 (1951).

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F. H. MATTSON AND L. W. BECK 125

5. Mattson, F. H., Benedict, J. H., Martin, J. B., and Beck, L. W., J. Nutr., 48, 335 (1952).

6. Borgstrom, B., Acta them. &and., 7. 557 (1953). 7. Martin, J. B., J. Am. Chem. Sot., 76,5483 (1953). 8. Lambert, M., and Neish, A. C., Canad. J. Res., Sect. B, 28.83 (1950). 9. Official and tentative methods of the American Oil Chemists’ Society, Chicago

(1954). 10. Cason, J., Sumrell, J., and Mitchell, R. S., J. Org. Chem., 16, 850 (1950). 11. Bush, M. T., and Densen, P. M., Anal. Chem., 20, 121 (1948). 12. Borgstrom, B., Biochim. et biophys. acta, 13, 491 (1954). 13. Schonheyder, F., and Volqvarta, K., Biochim. et biophys. acta, 8, 407 (1952). 14. Mattson, F. H., Benedict, J. H., and Beck, L. W., J. Nuts., 62,575 (1954). 15. Wills, E. D., Biochem. J., 67, 109 (1954). 16. Borgstrom, B., Biochim. et biophys. actu, 13, 149 (1954).

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F. H. Mattson and L. W. BeckLIPASE

TRIGLYCERIDES BY PANCREATIC THE DIGESTION IN VITRO OF

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