The Absorption of Fatty Acids by Functional Bovine Mammary Cells JOHN E. KINSELLA, Department of Food Science, Cornell University, Ithaca, New York 14850

ABSTRACT

Freshly dispersed bovine mammary cells rapidly absorbed long chain fatty acids from the culture medium. Differ- ences in the rates of absorption were observed, i.e., palmitic > stearic > oleic > myristic > linoleic acid. The pre- ponderance of the fat ty acids absorbed were esterified into triglycerides (>75%) and the remainder were mostly incorpo- rated into phospholipids. The cells secreted triglycerides into the culture medium. Of the phospholipid classes, phosphatidylcholine always contained most of the radioactivity in all experi- ments with labeled fat ty acids. These observations are related to the metabo- lism of mammary cells in vivo.

INTRODUCTION

The actual contribution of plasma free fat ty acids to the synthesis of ruminant milk fat, in vivo, had been equivocal until Annison et al. (1), using a combination of arteriovenous dif- ference measurements and isotope dilution techniques, showed that the lactating goat mammary gland absorbed substantial quantities of plasma free fat ty acids. Lauryssens et al. (2) reported that the perfused bovine mammary gland removed labeled stearic acid from the per- fusate and Patton and McCarthy (3) and Dimick et al. (4) showed that the goat mammary gland absorbed and metabolized free fat ty acids following their intramammary infusion. The lactating mammary gland also absorbs large quantities of free fat ty acids (3-4 g/100 ml secreted) which are liberated in the immediate vicinity of the gland by the action of l ipoprotein lipase on the triglycerides of the low density l ipoproteins ( 1,5-7).

While it is assumed that the secretory cells absorb and metabolize these free fat ty acids in vivo, it has not been actually demonstrated. Hence the present experiments were carried out to study the absorption and metabolism of free fatty acids and to examine the relative rates of absorption of different fa t ty acids by dispersed secretory cells obtained from lacta- ting bovine.

MATERIALS AND METHODS

The cells were prepared from fresh lactating t i s s u e o b t a i n e d f r o m H o l s t e i n cows immediately post mortem. The method of preparation, washing, and dispersal has been thoroughly described (8,9). The composit ion of the culture media and the incubation and harvesting methods have been reported (9). Following incubation, the cells (approximately l x l08 / t rea tment ) were separated from the cul- ture media by centrifugation (500 g for 10 min). The lipids were extracted from both the cells and culture media by the procedure of Folch et al. ( I0) . The lipid extracts were frac- t ionated by thin layer chromatography (TLC). The neutral lipid classes were separated on Silica Gel G plates, 250 /a thickness, using a solvent system of petroleum ether-diethyl ether-acetic acid (70:25:1 v/v/v). The phospho- lipid classes were fractionated by two-dimen- sional TLC (11). The various fractions were identified by co-chromatographing with stand- ard mixtures of known lipids (Applied Science, State College, Pa.). The various lipid classes were located using iodine vapor. After the lipid spots were marked, the iodine was evaporated by gently heating the plate to 60 C under a stream of nitrogen. The radioactivity in each spot was determined by transferring the silica gel spot and the adsorbed lipids to a scintilla- t ion vial, adding the scintillation fluors (2,5- d i p h e n y l o x a z o l e and 1,4-bis-(5-phenyloxa- zolyl-2)-benzene, Nuclear-Chicago, Chicago, Ill.) in toluene, and counting the sample in a (Packard TriCarb) liquid scintillation spectro- photometer . The recovery of radioactivity from these plates ranged from 77% to 103% and appropriate corrections were made for recovery and quenching effects when computing the final data. The efficiency of the scintillation counter (70%) was not considered in the calcu- lation of radioactive data.

The pure 1-14C fat ty acids (New England Nuclear, Boston, Mass.) were converted to their respective sodium salts (12) and introduced into the culture flasks at the initiation of each incubation. The TLC equipment and supplies were obtained from Brinkmann Instruments, Westbury, N.Y. and purified solvents (Mallin- ckrodt, St. Louis, Mo.) were used. Kodak No- Screen medical x-ray film (Kodak, Rochester,

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MAMMARY FATTY ACID ABSORPTION 893

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RESULTS

The initial series of experiments showed that the preponderance of the long chain fat ty acids absorbed by the cells were esterified. Most of these esters were triglycerides (70-80%) and the remainder were associated with the phospho- lipids and diglycerides (Table I). Significant quantities of ester lipids, predominantly trigly- cerides, appeared in the culture medium as the incubations progressed (Fig. 1). The radioactive triglycerides appeared as two and, occasionally three, discrete spots on the autoradiograms. These probably coincide with the triglycerides of different molecular weights which occur in mammary cells and milk (14).

Using the extent of esterification as an index of active absorption, the relative rates of uptake of various long chain fat ty acids, normally supplied to the lactating mammary gland, were measured and the results are summarized in Figure 2. The long chain saturated fat ty acids were absorbed to a greater extent than the unsaturated acids, particularly linoleic acid.

As demonstrated in the autoradiograms the adsorbed fat ty acids were predominantly ester- ified in triglycerides (70-80%) and most of the remainder was incorporated into the phospho- lipids (Table I). The phosphatidylcholine fractions contained most of the radioactivity associated with the phospholipids although p h o s p h a t i d y l i n o s i t o l , phosphatidylethanol- amine and phosphatidylserine were significantly labeled by all of the substrate fat ty acids studied.

DISCUSSION

These experiments demonstrated that the d i s p e r s e d secretory cells prepared from lactating bovine mammary tissue actively absorb and metabolize exogenous free fat ty acids. The cells showed a preference for satu- rated fatty acids, which is perhaps logical since the functional ruminant mammary gland normally absorbs large amounts of these, particularly palmitic and stearic acid, from the circulating plasma (1,4-6) even though signifi- cant quantities of unsaturated acids are also present. The data confirm the assumption that the secretory cells are responsible for fat ty acid absorption and metabolism in the lactating ruminant mammary gland.

The mode of absorption of the fat ty acids by mammary cells is unknown but conceivably

LIPIDS, VOL. 5, NO. I I

894 JOHN E. K I N S E L L A

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FIG. 2. The absorption of various fatty acids by freshly dispersed bovine mammary ceils. The quantity esterified indicates active absorption. C16:0, palmitic; C18:0, stearic; C18:I, oleic; C18:2, linoleic and C14:0, myristic acid.

FIG. 1. Thin layer autoradiogram of the radio- active lipids extracted from bovine mammary cells and culture media following incubation with 1-14C_sodium myristate for 4 and 8 hr. PL, phospholipids; MG, monoglycerides; DG, diglycerides; FA, free fatty acids; TG, triglycerides (two spots indicated).

the mechanism is similar to that proposed by Shapiro (15), i.e., it involves adsorption, acti- vation and absorption of the activated fatty acids. Esterification is the principal mechanism by which free fat ty acids are assimilated by tis- sues (15) and the present experimental obser- vations were consistent with such a mechanism because the preponderance of the fatty acids absorbed by the cells were esterified. Presum- ably the activation of these exogenous fat ty acids occured in the cell membrane since negligible quantities of free acids appeared in the cells and significantly, it has been reported (18) that the cell membrane fraction of the liver cell is the richest locale of long chain fat ty acid activating enzymes.

The rapid esterification of absorbed fatty acids has been observed in the mamary gland of the rabbit (16), goat (1), guinea pig (12), mouse (17), and by dispersed mammary ceils from the rat (Kinsella, unpublished data) and generally the triglycerides are the major lipid class

synthesized. Askew et al. (19) while studying glyceride synthesis by subcellular port ions of bovine mammary tissue, observed different rates of esterification for various fat ty acids (oleic ~ palmitic > stearic > linoleic). This o b s e r v a t i o n and the experimental data presented above may reflect different activation rates, acyl transferase activities and/or the relative availability of acyl acceptors in the mammary cell.

The bovine cells incorporated the various fat ty acids into the cellular phospholipid classes, indicating that the mammary cells can synthe- size these molecules at least partially (20). Characteristically, the phosphatidylcholine frac- tion contained the preponderance of the radio- activity in all of the experiments. Similar labeling patterns have been observed in vivo (3) and in vitro with other substrate precursors (20). These labeling patterns have been ration- alized by proposing an active metabolic role for phosphatidylcholine in milk lipid biosynthesis (21). Coincidentally, the pattern of absorption of the fatty acids by the mammary cells is quite similar to that observed by Van den Bosch et al. (22) in experiments with rat liver microsomes and using 2-acyl-3sn-glycerol-phosphorylcholine as the acyl acceptor. If such a mechanism was

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MAMMARY FATTY

opera t ing in the m a m m a r y cells it would account for the labeling of the phospha t idy l - choline; whe the r or no t it is a rou te to the synthesis of t r iglycerides (21) is the subject o f fur ther studies.

Finally, this s tudy demons t r a t e s tha t the bovine m a m m a r y cell can absorb and meta- bolize oleic and linoleic acid when these are p resen ted as free acids. The precise quant i ta t ive origin of these fa t ty acids in milk fat has no t been def ined. Significant quant i t ies o f the oleic acid are derived by desa tura t ion of stearic acid in the m a m m a r y gland (1,2,24) . However , dietary studies indicate tha t b o t h oleic and lin- oleic acids of milk fat can be derived f rom cir- culating plasma lipids (23).

ACKNOWLEDGMENTS

Technical assistance by Lorna Ulmer; supported by the National Science Foundation Grant GB-8430.

REFERENCES

1. Annison, E.F., J.L. Linzell, S. Fazakerley and B. Nichols, Biochem. J. 102:637 (1967).

2. Lauryssens, M., R. Verbeeke and G. Peeters, J. Lipid Res. 2:383 (1961).

3. Patton, S., and R.D. McCarthy, J. Dairy Sci. 46:396 (1963).

4. Dimick, P.S., R.D. McCarthy and S. Patton, Bio- chim. Biophys. Acta 116:159 (1966).

5. Barry, J.M., Biol-, Rev. 39:166 (1966).

ACID ABSORPTION 895

6. Bishop, C., T. Davies, R.F. Glascock and V.A. Welch, Biochem. J. 113:629 (1969).

7. Glascock, R.F., V.A. Welch, C. Bishop, E. Weight and R.C. Noble, Ibid. 98:149 (1966).

8. Ebner, K.E., C.R. Hoover, E.C. Hageman and B.L. Larson, Exptl. Cell Res. 23:373 (1961).

9. Kinsella, J.E., and R. McCarthy, Biochim. Biophys. Acta 164:518 (1968).

10. Folch, J., M. Lees and G.H. Sioane-Stanley, J. Biol. Chem. 226:497 (1957).

11. Parsons, J.P., and S. Patton, J. Lipid Res. 8:696 (1967).

12. McBride, D.W., and E.D. Korn, Ibid. 5:448 (1966).

13. Kinsella, J.E., Biochim. Biophys. Acta 210:28 (1970).

14. Blank, M.L., and O.S. Privett, J. Dairy Sci. 50:1194 (1967).

15. Shapiro, B., Ann. Rev. Biochem. 36:247 (1967). 16. Bulock, J.D., and G. Smith, Biochem. J. 96:495

(1965). 17. Stein, Y., and O. Stein, J. Cell Biol. 34:251

(1967). 18. Pande, S.U., and J.F. Meade, J. Biol. Chem.

243:352 (1968). 19. Askew, E.W., J.D. Benson, R.S. Emery and J.W.

Thomas, Fed. Proc. 28:623 (1969). 20. Kinsella, J.E., Biochim. Biophys. Acta 164:540

(1968). 21. Patton, S., R.O. Mumma and R.D. McCarthy, J.

Dairy Sci. 49:937 (1966). 22. Van Den Bosch, H., L.M. Van Golde, A.J. Slot-

boom and L.L. Van Deenen, Biochim. Biophys. Acta 152:694 (1968).

23. Moore, J.H., and W. Steele, Proc. Nutr. Soc. 27:66 (1968).

24. Kinsella, J.E., J. Dairy Sci., in press.

[Revised manuscr ip t received Sep t ember 28, 1970]

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