Glyceride glycerol utilization in triglyceride formation

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

93, 546-549 (1961)

Glyceride Glycerol Utilization in Triglyceride Formation I HERBERT

C. TIDWELL

AND JOHN

M.

JOHNSTON

From the Department o] Biochemistry, University o] Texas, Southwestern Medical School, Dallas, Texas

Received January 30, 1961 A comparison of the utilization of labeled fatty acids and glycerol, both free and combined, in the formation of glyeerides has been made employing an in vitro procedure with rat intestine. Small amounts of long-chain fatty acid glycerides were formed from glycerol-C 1~ or from monoaeetin or monopalmitin, both labeled in the glycerol moiety, in increasing amounts in the order named. This result was obtained although an appreciable hydrolysis of the monoglyeerides occurred during the incubation period. The partition of the glyeerides in the intestinal tissue suggested the absorption of some intact monoglycerides. However, the relative amounts of the various glyeerides found after the use of monopalmitin, fatty acid-labeled, resembled more those found after employing labeled free fatty acids than those after monopalmitin labeled in the glycerol moiety. Evidence suggests that most of the fat is completely hydrolyzed prior to absorption. INTRODUCTION Several investigators (i, 2) have presented evidence to suggest t h a t the appear-

ante of p a r t of the glyeeride glycerol, isolated from thoracic duct ehyle, is the result of intestinal absorption of glyeerides, since ingested free glycerol is not so incorporated to a significant extent (3, 4). An explanation for the latter finding offered b y Buell and Reiser (5) was the absence of a phosphorylating enzyme for glycerol in the intestinal mueosa. Monoglycerides have been proposed as the most likely precursors of the chyle glyceride glycerol of intestinal origin (1). This suggestion has recently been supported by Skipski et al. (6). T h e y reported t h a t monoglyeerides enter the mueosa without hydrolysis, since monoglyeerides were found in the recovered intestinal tissue lipids. These investigators have also suggested t h a t "the slight, or non-existent, reutilization of free glycerol-C 14 in the 1This investigation was supported in part by research grants from the Robert A. Welch Foundation, Houston, Texas, and the National Institute of Arthritis and Metabolic Diseases, U. S. Public Health Service, No. A-3108 and A-1522.

incorporation processes can best be explained by the low concentration of the tagged alcohol available at any particular site within the mueosal cells." Additional information has been sought regarding the origin of glyeeride glycerol by employing a recently developed in vitro procedure. I n this investigation free glyeerol-C 14, glycerol-labeled monopalmitin, or glycerol-labeled monoaeetin has been utilizd to determine the relative incorporation of the activity of these compounds into longchain f a t t y acid glyeeridcs ( L C F G ) . The latter water-soluble glyceride was employed to investigate the possibility t h a t monoglyeerides niight be split by the previously demonstrated intestinal enzyme (7), leaving the glycerol in a more available form, perhaps combined with the enzyme, and thus enhance its incorporation into L C F G . EXPERIMENTAL SYNTHESIS OF LABELED ~OMPOUNDS The monopalmitin, labeled with C ~ in the glycerol moiety, was prepared as previously described (7). Monoacetin was similarly prepared from glyc-

erol-C 14 and acetic acid by the following proce546

GLYCERIDE GLYCEROL UTILIZATION dure. To 0.2 ml. glycerol containing 20 ~e. glycerol1-C ~4 was added 0.2 ml. of glacial acetic acid. The mixture was stirred at 110°C. for 1 hr. under an atmosphere of C0~. After cooling, the residue was extracted three times with benzene and three times with chloroform. Following the addition of another 0.2 ml. acetic acid to the remaining residue, the entire procedure was repeated. The chloroform extracts were combined, the solvent was removed, and 4 ml. water was added. The aqueous solution was re-extracted with benzene and then with ehloroform, to remove most of the higher glycerides. The monoacetin and remaining glycerol were recovered by removing the water at 35°C. under reduced pressure. Repeated separations with chloroform freed the monoacetin from any remaining glycerol and gave a preparation containing only aeetins of which 75-90% was monoacetin. This result was evidenced by the separation of known mixtures in a similar manner. The radioactivity of the monoacetin preparation was approximately 1500 eounts/min./mg. S O L U T I O N AND

TISSUE P R E P A R A T I O N S

The labeled monoacetin preparation was added to a f a t t y acid-albumin solution in the amount of 2 mg./ml. I n the preparation of the labeled glycerol solution, the monoacetin was replaced with molar-equivalent amounts of glycerol-C 1. and acetic acid. The preparation of all solutions for incubation with the intestinal segments, the incubation procedures, and the radioactivity determinations on the recovered labeled compounds were carried out as previously described (7). After the addition of carrier substances, all extractions of the mueosal and serosal solutions and intestinal tissues were

547

made with hot alcohol and then with ether in order to include the water-soluble glycerol and monoacetin as well as the lipids. The combined solvents from each extraction were removed, and the residue was separated into benzene- and water-soluble fractions. The benzene fraction was further separated into the various glycerides and free f a t t y acids (8). Radioactivity measurements were made on each fraction. RESULTS AND DISCUSSION

The distribution of the activity, originally in the mucosal solution, in the various compartments is shown in Table I. The last two lines are data taken from a previous report (7) to permit a comparison of results. The data indicate that in this in vitro preparation small amounts of glycerol could be incorporated into LCFG, whether the glycerol was uncombined or in monoesters of long- or short-chain fatty acids. Although the amount of LCFG formed was small, in all three compartments there was a consistently greater incorporation of the labeled glycerol from monoacetin into LCFG than from the free glycerol. This increase in incorporation of the glycerol from monoacetin might have been larger if an appreciable hydrolysis of the ester in the mueosal solution had not occurred. The hydrolysis of 55% of the monoacetin after 1 hr. and 62% after 2 hr. incubation left predominantly free glycerol instead of monoacetin available for absorption. However, the consist-

TABLE

I

D I S T R I B U T I O N OF ACTIVITY IN VARIOUS COMPARTMENTS AFTER INCUBATION Mucosal solution

Preparations a

No. sacs used

PA MA* Gly* MP*

6 10 10 4

PA MP

12 12

Hydrolysis Original total during activity on incubation LCFG

Intestinal tissue

Serosal solution

Per cent of total original activity As LCFG formed

As LCFG/ g. each

As LCFG/ ml. each

Relative amounts of LCFG

%

%

-62 18

2.4 0.8 0.6 0.7

15.5 1.7 0.7 5.7

104.6 4.5 3.4 44.6

5.30 0.80 0.66 0.81

100 15 12 15

--

2.1

19.0 5.0

40.4

5.40 1.10

20

--

20

PA, MA, Gly, and M P refer to f a t t y acids, m o n o a c e t i n , glycerol, and m o n o p a l m i t i n respectively° Those w i t h a s t e r i s k i n d i c a t e label with C 14 in glycerol moiety; others c o n t a i n e d palmitic aeid-l-C 14.

5~i8

TIDWELL AND JOHNSTON

eney of the finding in each compartment supports the suggestion that the glycerol from monoaeetin was in a more available form for LCFG formation. One additional explanation of an increased availability might be the possibility of monoacetin undergoing transesterification with the free palmitic acid (9). Small but definite amounts of labeled LCFG were present after employing monoacetin, monopalmitin, or free glycerol, all labeled with glycerol-C14. This would suggest that there must be some mechanism whereby at least limited amounts of free glycerol can be incorporated into triglycerides. Favarger and Collet (10) have reported some incorporation of glycerol into glycerides in the intact animal. The finding of labeled glyeerides in the serosal solutions from the labeled free glycerol, in contrast to findings in intact animals (2, 3), supports the suggestion of Skipski et al. (6) regarding the availability of glycerol. In the in vitro system of this study, the free glycerol or that hydrolyzed from the glyeerides could not be transported away from the absorptive area and might thus have been present in a more available form and in sufficient concentrations at particular sites within the mucosal ceils to permit incorporation into the labeled glyeeride. This might be analogous to in vivo utilization of part of the glycerol gradually freed by lipolysis in contrast to ingested free glycerol which may be rapidly absorbed and transported away via the

portal circulation. If the absorption of glycerol, given as such, Should happen to be complete in the upper part of the intestinal tract, it would not be present at the site where the fatty acids were freed and hence could not combine with them to form LCFG. The monoglycerides, resulting from the action of pancreatic lipase are largely, if not exclusively, in the B-form (11), while the one employed in this study was undoubtedly primarily the a-monoglyeeride. It is possible that the B-form is more readily utilized for glyeeride formation than the a-configuration, but the ratio of the two has been shown to change from that of largely B-isomer in early digestion to equilibrium proportions of 9:1 of ~:fi-forms at the end of 1 hr. in the intestinal tract (11). This intereonversion would seem to render the form to be employed less important since both forms would be available for utilization. The data in Table I would indicate the possible absorption of a small but significant portion of unhydrolyzed monoglyceride. Although the glyeeroI of the monoaeetin was used in LCFG formation to a greater extent than that of the free glycerol, hydrolysis or transesterifieation (9) must have occurred to permit formation of these glycerides. Also, the possible absorption of intact monopalmitin is indicated by the appearance of approximately the same total activity in the glyeeride fraction of the intestinal tissue regardless of whether the

TABLE II PARTITION OF GLYCERIDES RECOVERED FROM INTESTINAL T I S S U E AFTER INCUBATION WITK VARIOUS PREPARATIONS Monoglycerides Preparationsa

PA Gly* MP* MP PA

Diglycerides

Triglycerides

Total glycerides of original activity

Of original total activity

Of total glycerides

Of original total activity

Of total glycerldes

Of original total activity

Of total glycerides

% 0.7 0.1

% 5.1 22.5 23.0

% 2.2 0.5 2.5

% 16.4 43.7 46.0

% 10.4 0.3 1.6

% 78.5 33.8 30.1

% 13. 3 0.9 5.3

16.0

1.3 3.5

26.0

2.9 15.5

58.0 81.6

5.0 19.0

1.2

0.8 0.0 See footnote to Table I.

18.4

GLYCERIDE GLYCEROL UTILIZATION

549

label was present in the glycerol or fatty free fatty acids present. I f only one-third acid portion of the monoglyceride. The ap- as much activity were found in serosal glyepearance of less activity in this tissue after erides when fatty acid-labeled monopaluse of glycerol-labeled monoaeetin may pos- mitin was used instead of free fatty acids sibly be explained by the approximately (Table I), and i f all of this were absorbed three times as much hydrolysis of the mono- as monoglyeeride from the partial hydrolyaeetin as the monopalmitin in the mueosal sis of triglyeerides, then approximately 90% solution. However, ik is to be noted that the of all the original glyeeride fatty acids activity in the LCFG was greater in the would have been absorbed as free fatty serosal solution when the monopalmitin was acids. One cannot escape the conclusion that labeled in the fatty acid portion rather than at least the major mechanism involved is the glycerol moiety. Furthermore, the dis- that of the absorption of free fatty acids tribution of activity (Table II) obtained with only a limited absorption of intact from a fraetionation of the glycerides of the glyeerides. intestinal tissue after use of the monoglycACKNOWLEDGMENTS eride labeled in the fatty acid fraction definitely more resembles that after the use of The authors gratefully acknowledge the technilabeled free fatty acids than that after the cal assistance of Miss Genevieve Boerner, Mr. J. monoglyceride-containing labeled glycerol Lon Pope and Mrs. Lorraine Johnson. --58 and 78% as compared with 30% in triglycerides and 26 and 16% as against 46% REFERENCES in the diglyeerides. These in vitro findings 1. REISER,R., ANDWILLIAMS,M. C., J. Biol. Chem. are not in accord with the suggestion of an 202, 815 (1953). appreciable absorption of intact monoglye2. BLOOMSTRAND, R., BORGSTR()M, B., AND DAI:ILerides for conversion into triglyeerides RACK,O., Proc. Soc. Exptl. Biol. Med. 102, which later appear in the chyle. 2O4 (1959). In the absence of a known mechanism in 3. REISER, R., BRYSON, M. J., CARR, M. J., AND the mueosa for the formation of triglyeerKUIKEI',T, K. A., J. Biol. Chem. 194, 131 ides from monoglyeerides, the presence of a (1952). 4. BERNHARD, K., WAGNER, I-I., Ah'D RITZEL, G., lipolytie enzyme within the intestinal muHelv. Chim. Acta 35, 1404 (1952). eosa could account for results obtained dur5. BUELL, G. C., AND REISER, R., J. Biol. Chem. ing active absorption. Hydrolysis of the 234, 217 (1959). monoglyceride within the mucosa could free 6. SKIPSKI, ~. P., MOREHOUSE, M. G., AND I)EUEL, fatty acids to form triglyeerides through the It. J., JR., Arch. Biochem. Biophys. 81, 93 known mechanism involving a-glyeerophos(1959). phate (12-14). The liberated glycerol might 7. TIDWELL, I-I. C.t AND JOHNSTON, J. M., Arch. be in a more available form and in such conBiochem. Biophys. 89~ 79 (1960). centrations as required for its utilization to 8. BORGSTn6~, B., Acta Physiol. Scan& 25, 111 form glyeerides. (1952) ; 30, 231 (1954). 9. AHRENS, E. H., AND BORGSTRSM, B., J. Biol. Nevertheless, it must be remembered that Chem. 219, 665 (1956). the incorporation of activity into glycerides from free fatty acids is approximately 4-5 10. FAVARGER,P., AND COLLET, R. A., Helv. Physiol. et Pharmacol. Acta 8, C15 (1950). times greater than that from the fatty acids 11. MATTS0N, F. ]=I., A~D BECK, L. W., J. Biol. of monoglyeerides in this in vitro preparaChem. 214, 115 (1955); 219, 735 (1956). tion. Also the amount of activity appearing 12. DAWSON,A. M., A~D ISSELBACHER,K. J., J. Clin. in the intestinal lipids amounted to less Invest. 39, 150 (1960). than 6% of the total original activity when 13. CLARK, B., AND HfJBSCHER, G., Nature 185, 35 approximately 20% of the monopalmitin (1960). had been hydrolyzed. If fatty acids are se- 14. Jo~STO~, J. M., A~'DBEARDEN,J. H., Arch. Biolectively and more rapidly absorbed (15), chem. Biophys. 90, 57 (1960). it would appear reasonable to assume that 15. BORGSTRSM,B., Acta Physiol. Scan& 25, 291 (1952). most of the glyceride was formed from the