[Agr. Biol. Chem., Vol. 28, No. 4, p. 193•`200 1964]

Glyceride Structure and Biosynthesis of Natural

Part I. Specificity for of Pancreatic Lipase

By Osanlu HIRAYAMA Collegeof Agriculture,Kyoto Prefectural University, Kyoto ReceivedSeptember 30, 1963

In order to study specificity of pancreatic lipase, a number of synthetic triglycerides were hydrolyzed by the enzyme under an improved condition. The proportions of isomers of the derived mono- and , and the compositions of the derived free acids and were de termined. The hydrolyzing rate of fatty acids in depended on the position esterified in the , carbon number of the acid, and structure of the . Positional specificity of the enzyme was markedly displayed for symmetrical triglycerides composed of long chain acids, but at somewhat lower rate for glycerides containing short chain or highly unsaturated acids.

INTRODUCTION •@ acids esterified in triglycerides. The glyceride structure of natural fats and In the present paper, an effect of carbon- has been studied over a number of years chain length and unsaturation degree of fatty by use of the technique separating triglyce acids in triglycerides upon the specificity of

rides or the method of pancreatic lipase hy pancreatic lipase was investigated, and the

drolysis. The latter enzymatic procedure, positional specificities were also examined. particularly, was used in an extensive study These studies were carried out to demon by Savaryl`31 and Mattson4). They demon- strate in detail glyceride structure in animal strated that unsaturated fatty acids pre- and vegetable fats, and to follow a study on dominated on the 2 position of triglycerides the biosynthetic mechanism of glyceride from a large number of animal and vegetable structure.

fats. This method of enzymatic hydrolysis is MATERIALS AND METHODS based on the specificity of pancreatic lipase Syntheses of Triglycerides. A number of synthetic for hydrolyzing the linkages at the 1 triglycerides were obtained by the method as described and 3 positions of triglycerides and on an in Fig. 1. Monoacid triglycerides were synthesized by assumption that the enzyme did not show a the procedure (1)6), and diacid triglycerides by the selectivity for the nature of fatty acids esteri procedure (2)7) (3) 8) and (4) 9) These products fied at these position. However, a recent were passed through silica gel column according to a modified technique of the procedure described by reports5) indicated that positional specificity Quinlin10), and the isolated triglycerides were re- of the enzyme was influenced by short chain ) D.H. Wheeler, R.W. Ricmenchneider and C.E. Sando, J. Biol. Chem., 132, 687 (1940). 1) P. Savary and P. Desnuelle, Biochim. Biophys. Ada, 21, 349 7) B. F. Daubert, H.H. Fricke, and H.E. Longenecker, J. Am. Chem. (1956). Soc., 65, 2142 (1943). 2) P. Savary, J . Flanry and P. Desnuelle, ibid., 24, 414 (1957). 8) B.M. Craig, W.O. Lundberg, and W.F. Geddes, J, Am. Che 3) P. Savary and P. Desnncllc, ibid., 31, 26 (1959). mists' Soc., 29, 169 (1952). 4) F.11. Mattson and L.W. Beck, J. Biol. Chem., 214, 115 (1955); 9) B.F. Stimmel and H.G. king, J. Am. Chem. Soc., 56, 1724 (1934). 219, 735 (1956) . 10) P. Quinlin and H.J. (Weiser, Jr., J. Am. Oil Chemists' Soc., 35, 5) P. Savary, et al., Bull. Soc. Chim. Biol., 43, 581 (1961). 325 (1958).

6 194 Osamu HIRAYAMA

FIG. 1. Synthetic Procedures of Triglycerides. M : A mixture of saturated even numbered acids (Cs-Cs.) or a mixture of saturated even and odd numbered acids (C7, C10, C18, C18, C19). MCI : The chlorides of fatty acid mixtures.

TABLE I. MELTING POINTS AND IODINE VALUES OF SYNTHETIC TRIGLYCERIDES

a) Number : Carbon No. of fatty acids, (') : Number of unsaturation in fatty acids. b) No. of synthetic procedure described in Fig. 1. crystallized. Melting points and iodine values of the of two mixtures of diacid triglycerides. One mixture triglycerides were shown in Table I. These values, (Diacid Mixture I) was prepared by the the fatty acid compositions, and the paper chromato reaction of 2,3-diolein12) with chlorides of equi-molar grams11) indicated that the synthetic triglycerides were mixture of saturated even numbered acids (C6-C24), considerably pure. and another mixture (Diacid Triglyceride Mixture II) The procedure (5) was employed for preparation 12) prepared by the procedure as described by Buchnea (D, Buchnea, 11) O. Hirayama and Y. Inouye, J. Agr. Chem. Soc. Japan, 35, 372 Fette. Seifen. Anstrichmittel, 64, 887 (1963)). Glyceride Structure and Biosynthesis of Natural Fats. Part I 195 was obtained by acylation of 2,3-diolein12) with chlo homogenizer obtained from Nippon Seiki Seisakusyo. rides of equi-molar mixture of saturated even and The incubation mixture consisted of 6ml tris-buffer odd numbered fatty acids (C7,C10, C13, C16 and (0,5M, pH 7.5), 0.5ml of 45% solution of calcium C19). These products were refined on silica gel chloride, 2ml of 1% aq. solution of deoxycholate, 3ml column as described above. The fatty acid composi of suspension of 100mg lipase in tris-buffer (0.5M, tions and the paper chromatograms12) of the products pH 8.0), and 0.5g of triglyceride. This scale of were shown in Tables IV and V, and in Fig. 2. reaction mixture was changed corresponding to the A number of natural fats and oils refined were also amounts of substrates only when mixtures of glycerides employed in the present study. as described later were applied to the incubation. Under these condition, the lipase hydlolyzed about Enzymatic Hydrolysis. The "lipase" preparation 60% of total ester linkages in most natural fats and was purchased from Tokyo Kasei Ind. Co. The con dition of digestion employed in the present investiga oils in 10•`15min. tion was essentially same to those described by Mattson At the end of incubation period, 5ml of a mixture et al.13), except that, during the incubation period, of N HCI-95% EtOH (3:5, v/v) was added and hydro the mixture was continuously homogenized with a lyzates were extracted with diethyl ether or chloro form. In the enzymatic hydrolysis for estimating the

proportions of mono- or isomers, the addition of the HCl-EtOH mixture after the reaction was omitted to prevent the isomerization.

FIG 2. Separation of Lower Triglycerides and Fatty Acids by Paper Chromatography. Solvent system : Stationary phase, kerosene ; mobile phase, methyl acetate-methanol-acetic acid-kerosene. (2 : 10 :2: 2, v/v) Color reagent : Rhodamine B. FIG. 3. Separation of Diglyceride Isomers by Thin- PC value: Total carbon No. of three component acids in triglyce Layer Chromatography. ride. Adsorbent: Silica gel. Sample Solvent system: Petroleum benzine-diethyl ether-acetone. S, pure synthetic triglycerides ; IG, Diacid Triglyceride Mixture I Color reagent: 10% ethanolic solution of phosphomolibdic acid. (described in Text); IF, free acid mixture isolated from products of Sample. of the enzymatic hydrolysis of IG ; IIG, Diacid Triglyceride Mixture S; monopalmitin, , 1,2-dipalmitin, 1,3-dipalmitin, II (described in text); IIF, free acid mixture isolated from products and ; A, products obtained by the enzymatic hydrolysis ofthe enzymatic hydrolysis of IIG. of 2-oleo-dipalmitin ; B, products obtained by the enzymatic hy- 13) F.H. Mattson and R.A. Volpenhein, J. Research, 2, 511 drolysis of I-palmito-diolein. MG, ; FA, fatty acid; (1961). DG, diglyceride; TG, triglyceride. 196 Osamu HIRAYAMA

Analytical Methods. The products of enzymatic drolysis: Monoacid Triglyceride Mixture I hydrolysis were extracted three times with diethyl (about equi-molar mixture of tristearin, tri ether. The ether solution was passed through a col palmitin, , trilaurin, tricaprin, tri umn of Amberlite IRA-400, and the free fatty acid caprylin, and tricaproin), Monoacid Trigly fraction and the partial glyceride fraction were ob tained. The glyceride fraction was subjected to ceride Mixture II (about equi-molar mixture column chromatography of silica gel as described of tristearin, tripalmitin, trimyristin, , above, to separate into mono-, di-, and triglyceride trilinolein, and trilinolenin), and Diacid Tri fraction. glyceride Mixture I and II (as described The diglyceride isomers in the products were quali above). The hydrolysates were separated tatively estimated by thin-layer chromatography, in into 4 fractions of free acids, mono-, di-, and which glycerides were run on silica gel* plate with triglycerides, and the fatty acid compositions petroleum ether-diethyl ether-acetone (70:20:3, v/v) of the free acid fractions and the mono as the developing solvent system. The monoglyceride isomers were determined by periodic acid oxidation glyceride fractions were determined. The before and after perchloric acid isomerization14). hydrolysis rate of individual acid was calcu Fatty acid compositions in fractions of free acids, lated as percentage of free acid to total of the mono-, di- or triglycerides were determined by the acid esterified in sample glycerides. These technique of paper chromatography described in pre results were presented in Tables II, III, IV vious paper15) and V. RESULTS Table II showed that lower triglycerides For the purpose of confirming a difference such as tricaprylin and tricaproin were hydro of hydrolysis rate among various fatty acids lyzed at a slow rate in the biginning of the esterified at the same position of glycerol, reaction and at a rapid rate with progress the following mixtures of synthetic triglyce of the reaction. The results (Table III) rides were applied to the enzymatic hy obtained from Monoacid Triglyceride Mix-

TABLE II. COMPARISON OF HYDROLYZING RATE IN FATTY ACIDS OF DIFFERENT CARBON NUMBER BY LIPASE HYDROLYSIS OF MONOACID TRIGLYCERIDE MIXTURE la)

a) A mixture of trisetarin, tripalmintin, trimyristin, trilaurin, tricaprirt, tricaprylin, and tricaproin . b) % of total free acids to total fatty acid esterified in sample (average rate of hydrolysis) . c) % of free fatty acid to total of the corresponding fatty acid esterified in sample.

* Silica-Rider obtained from Daiiti Kagaku Yakuhin Co . was used. 14) J.B. Martin, J. Am. Ch±m. Soc., 75, 5483 (1953); L. Hartman, J. Am. Oil Chemists' Soc., 39, 126 (1962). 15) O. Hirayama and Y. Inouye, J. Agr. Chem. Soc. Japan, 35, 135 (1961). Glyceride Structure and Biosynthesis of Natural Fats. Part I 197

TABLE III. COMPARISON OF HYDROLYZING RATE TABLE IV. COMPARISON OF HYDROLYZING RATE IN FATTY ACIDS OF DIFFERENT UNSATURATION IN FATTY ACIDS OF DIFFERENT CARBON NUM DEGREE BY LIPASE HYDROLYSIS OF MONOACID BER BY LIPASE HYDROLYSIS OF DIACID TRIGLY TRIGLYCERIDE MIXTURE IIa) CERIDE MIXTURE la)

a) A mixture of tristearin, tripalmitin, trimyristin, triolein, trilino lein, and trilinolenin. b), c) The same as described in Table II.

Lure II indicated that there was considerable a) Described in text. difference in the rate of hydrolysis between b), c) The same as described in Table II. tristearin and triolein, though triolcin, tri linolein, and trilinolenin gave about similar somewhat slower rates than those of even rate. numbered acids in 18% lipolysis period. From the experiment on diacid triglyce In Tables VI and VII, positional specificity rides synthesized from 1,2-diolein, it was ob of the lipase was investigated as follows. Each served that fatty acids (C6•`C24) esterified in of pure synthetic triglycerides was hydrolyzed the outside position of glycerol gave higher by the lipase, and the products were extracted rate of hydrolysis with decreasing carbon with chloroform. One part of the products number (in Table IV). In the period of was employed for determination of the pro lower lipolysis, however, longer chain acids portions of mono- and diglyceride isomers were liberated somewhat more rapidly than formed. Another part was fractionated, and shorter ones (in Table V). Table V also the free acid and monoglyceride fraction iso suggested that odd numbered acids gave lated were subjected to the analyses of fatty

TABLE V. COMPARISON OF HYDROLYZING RATE IN FATTY ACIDS OF DIFFERENT CARBON NUMBER LIPASE HYDROLYSIS OF DIACID TRIGLYCERIDE MIXTURE IIa)

a) Described in text. b), c) The same as described in Table II. 198 Osamu HIRAYAMA

TABLE VI. PROPORTIONS OF MONOGLYCERIDE ISOMERS DERIVED FROM SYNTHETIC AND NATURAL TRIGLYCERIDES BY LIPASE HYDROLYSIS

a) Number: Carbon No. of fatty acids, ('); Number of unsaturation in fatty acids. b) Mol. % in total monoglycerides.

TABLE VII. FATTY ACID COMPOSITIONS IN Two FRACTIONS OF FREE FATTY ACIDS AND MONOGLYCERIDES DERIVED FROM KNOWN TRIGLYCERIDES BY LIPASE

a) Number: Carbon No. of fatty acids, ('): Number of unsaturation in fatty acids . b) Mol. % in total monoglycerides. acid compositions. Values in Table VI vation by thin-layer chromatography showed showed that the proportions of 1-isomer in that the diglycerides formed by the lipase the monoglyceride formed increased with were nearly composed of only 2-isomer , and shortening carbon-chain length or with in very small spots of 1,3-isomer appeared ap crease of unsaturation degree of the compo proximately corresponding to the proportions nent acids in monoacid triglyceride series, of 1-monoglyceride in the same product as and that symmetrical glycerides gave higher shown in Fig. 2 as an example. Table VII proportions of 2-isomer than those of unsym suggested that most free acids came from the metrical ones in monooleo-dipalmitin and 1, 3 positions of original triglycerides, and monopalmito-dicaprylin. Qualitative obser most acids in the monoglyceride fractions Glyceride Structure and Biosynthessi of Natural Fats. Part I 193 were those from the 2 position. In the Positional specificity of the lipase seems to be suggested more precisely by the values of glyceride containing in 2 posi tion, however, considerable amount of cap proportions in mono- and diglyceride isomers rylic acid than expected was liberated, and formed than by the fatty acid compositions the formed monoglyceride contained less in the derived free acids and monoglycerides, amount of the acid. These unusual results because the latter values contained small er were also indicated by the proportions of ror caused by a few process of treatment. monoglyceride isomers formed. From the results in Table VI, specific hydro lyses for outer chains of various triglycerides DISCUSSION by the lipase are assumed to occur easily Pancreatic lipase split fatty acid in according to the order as follows: higher the same position of triglycerides at different triglyceride>lower triglyceride, lowly unsatu rate with the nature of the acids. The hydro rated triglyceride>highly unsaturated trigly lyzing rates were considerably associated with ceride, symmertical triglyceride>unsymmet structure of triglycerides, glyceride composi rical triglyceride. The question as to whether tion of reaction mixture, and time course of the relation for the positional specificity enzymatic reaction. Particularly, high melt depends on any factor can not be answered ing longer acids were liberated smoothly by here satisfactorily, but the isomerization of the lipase only when these acids were esteri mono- and diglyceride and the difference of fied in low melting mixed glycerides or when hydrolysis rate on the basis of the nature of their monoacid triglycerides were digested fatty acids might be considered as affecting with other low melting glycerides. Short factors. chain acids also appeared to be hydrolyzed Especially the isomerization of inner chain more rapidly in the mixed-glycerides contain to the outside positions in glycerol molecule ing longer chain acids than in their monoacid may occur very easily as suggested in the glycerides. experiment reported by Mattson and Vol Thus, the hydrolysis rate of each acid penhein16). among Tables II, III, IV, and V differs in the Thus, plausible interpretation for the high same condition of digestion, influenced by er contents of 1-monoglycerides formed from the above factors and others. However, the lower triglycerides or unsaturated triglyce following common relation in the rate of rides (in Table VI) and the higher lipolysis hydrolysis among various acids are observed. of lower triglycerides in latter period of re In initial period of enzymatic reaction shorter action (in Table II) may be that shorter or chain acids are liberated more slowly than unsaturated acids in the formed 2-monogly longer ones, and then they increase the rela cerides and 1,2-diglycerides migrate from in tive hydrolysis rate with time course of reac ner position to outer positions in glyceride tion, and in latter period give higher rate molecules, and then the acids in outer posi with shortening carbon-chain length of acids tions arc attacked by the lipase, when shorter (C6•`C24). The effect of unsaturation in chains are removed more rapidly than longer fatty acids are markedly shown between ones. stearic and oleic acid esterified in mono- or The lipase preparation used in the present diacid tirglycerides, but not remarkable dif study is not sufficiently pure. Therefore, all ference was observed among three acids of 16) r.H. Mattson and R.A. Volpenhein, J. Lipid Research, 3, 281 oleic, linoleic, and linolenic. (1962). 200 Osamu HIRAYAMA results obtained above might be not precisely shown by the proportions of the derived corresponding to the properties of pancreatic monoglyceride isomers in Table VI. But, in lipase. However, practically the specificity the case of natural glycerides containing a observed could be presumed as those of the large quantity of short chain acids or highly enzyme. Under the condition of digestion unsaturated acids, the technique must be employed here, positional specificity of pan cautiously applied, in which the glycerides creatic lipase can be effectively used as an should be digested to a low average rate of improved method for determination of gly hydrolysis. ceride structure in natural fats and oils as