P O S T A B S O R P T I V E D I S T R I B U T I O N OF 14 C - L A B E L E D F A T T Y A C I D S IN SHEEP 1,2 D. A. Cramer and L. G. Miller3

system, in chickens, with similar inhibition studies. This study was designed to trace the digestion, absorption and deposition of 14 C-labeled lipids in the sheep in order to observe in situ the sequence of alterations that occur in the metabolism of lipids in sheep.

SUMMARY

Four mature ewes were administered via rumen fistula l a C-labeled triglycerides diluted in a bile-lipid-ethanol mixture. Two of the ewes were treated with tristearin-1J 4 C and the other two with trilinolein-1-I 4 C. Three days later the animals were slaughtered. Fatty acid and radioactive compositions of various tissue lipids were determined. Differences in fatty acid composition were observed among rumen ingesta, intestinal contents, liver and other tissue lipids. (Key Words: Sheep, Fatty Acid Composition, Radioactive Triglycerides.)

EXPERIMENTAL PROCEDURE

INTRODUCTION

Reiser and Reddy (1956) and Shorland et al. (1957) reported that there is more C18 saturated fatty acid (stearic acid) than C18 unsaturated fatty acids in rumen contents regardless of the C18 unsaturated fatty acid content of dietary feedstuffs. However, average values taken from published literature (Cramer et al., 1967; Thrall and Cramer, 1971) for the fatty acid composition of ruminant depot fats indicate that there is more C18 unsaturated fatty acids than stearic acid deposited in adipose tissue. Specific changes in dietary lipids probably take place prior to absorption in the rumen and gut. In addition, changes could take place in the blood, liver, other organs, or in adipose tissue as the fat is being deposited. Raju and Reiser (1967) demonstrated the presence of a stearic acid desaturase system in rats via inhibition studies with cyclopropene fatty acids. Allen et al. (1967) also demonstrated this

1Approved for publication as Scientific Series Paper No. 1643, of the Colorado State University Experiment Station. 2Contributed to Western Regional Project W-61. This paper is part of a thesis presented in partial fulfillment of the requirements for the Ph.D degree. SPresent address: Carnation Research Laboratories, Van Nuys, California.

Four mature Columbia ewes were fitted with ruminal fistulae and fed a 75% alfalfa (IRN 1-00-023), 15% milo (IRN 5-04-605) and 10% barley (IRN 4-00-549) pelleted ration twice daily. After allowing 1 week to condition the ewes to the ration, two of the ewes were dosed with tristearin-l-14C (9.7 mc/mM) and the other two at comparable times with trilinolein1 ) 4 C (30 mc/mM). The total dose administered to each ewe was about .5 me. The labeled glycerides were diluted to 50 ml with bile and carrier lipid (tallow and oleic acid) and 95% ethanol. This dilution was introduced into the ventral portion of the rumen through the fistula. The 14C.labeled triglycerides were obtained from Volk Radiochemical Co., Skokie, Illinois and were guaranteed to have 99% radiopurity as based on reverse phase paper chromatography and thin layer chromatography assay procedures. At the end of the trial period of 3 days the ewes were slaughtered and within 20 min after slaughtering the abdominal cavities of the animals were exposed. Muscle, adipose tissue and liver were immediately frozen. Total whole blood was collected and weighed. Samples of plasma were frozen and stored at - 2 0 C. The rumen-reticulum, omasum, abomasum, duodenum, jejunum, ileum, and upper, middle and lower colon sections were stripped of their contents and the contents were stored at - 2 0 C for subsequent analyses. Lipids were extracted from all tissue samples with a solvent mixture of isopropyl alcoholheptane-4 N H2SO4 (4:1:0.1) according to

884 JOURNAL OF ANIMAL SCIENCE, Vol. 43, No. 4 (1976)

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Colorado State University, Fort Collins 8 0 5 2 3

DISTRIBUTION OF ~~C-LABELED FATTY ACIDS IN SHEEP

RESULTS A N D DISCUSSION

As may be observed in table 1, lipids undergo many alterations in fatty acid composition from the time they are consumed until

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Dole and Meinertz (1960). This method yeas selected because autoradiographs of thin layer plates of lipids from the various compartments of the gastro-intestinal tract showed that the lipids were over 95% free fatty acids. The same method was used on all samples for consistency. The aqueous phases were washed with heptane and the organic phases with distilled water several times. F a t t y acids were esterified as outlined by Cramer and Marchello (1964) and modified by Miller (1967). An Aerograph A-700 Autoprep gas-liquid chromatograph equipped with a turntable was used for separation and collection of fatty acids. Chromatographic operating conditions were as follows: Oven temperature, 230 C~ injection block temperature, 330 C; collector temperature, 230 C; carrier gas (helium) flow, 80 ml/min; column, 610 cm • .635 cm (O.D.), 15% polydiethylene-glycol succinate on Chromosorb P, 80-100 mesh; detection system, thermal conductivity; method of quantitation, Model 201 Disc Integrator, Disc Instruments, I n c . About 10 /~s of each sample were injected into the chromatograph and collected as seven individual fractions (eight fractions from liver lipids) as they were eluted from the column. In some of the fractions fatty acids were collected as groups to avoid contamination with ~4C labeled fatty acids by possible overlap of eluents. A test of identity of the collected effluents was based on retention times of fatty acid standards obtained from The Hormel Institute 9 The unlabeled total fatty acids in table 1 were grouped the same as the radioactive fatty acids so that the distribution of radioactivity could be compared to the percentage composition of the total fatty acids o f each tissue lipid 9 Each fraction was counted for a total of 20 rain with a Beckman Model 1650 liquid scintillation system. The scintillation mixture consisted of 4 g PPO (2,5-diphenyloxazole) and .1 g dimethyl-POPOP 1,4-bis (2-(4-methyl-5-phenyloxazolyl)) benzene made up to one liter with toluene. Corrections for quench and recovery o f the samples from the gas-liquid chromatograph were made. Calculations were obtained as relative percentage of disintegrations in the seven fractions.

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DISTRIBUTION OF 14 C-LABELED FATTY ACIDS IN SHEEP

ly, of the total fatty acids of subcutaneous fat. The fatty acids making up depot lipids are probably a composite of endogenous fatty acids and dietary fatty acids that are desaturated in the adipose tissues. An interesting shift occurred in the radioactivity of the lipids in the 14 C-tristearin dosed sheep in table 2. In the various tissues, 80 to 90% of the radioactivity was in stearic acid except for blood, adipose and muscle tissue lipids. In the adipose tissue it appeared that about two-thirds of the labeled stearate had been desaturated to oleate. In the muscle tissue about the same amount of stearate was desaturated to oleate and in addition about one-third of the oleate was further desaturated. In speculating that desaturation occurred, the possibility of preferential deposition of unsaturated fatty acids must not be overlooked. It would be interesting to determine how much of the labeled C18:2 was actually linoleic acid because according to Mead (1961) and Schroepfer and Bloch (1965) the major C18:2 acid resulting from desaturation of oleic acid would be A 6'9-OctadecadienOic acid rather than linoleic acid (A 9,12.octadecadienoic acid). Such a determination would give good evidence of the amount of desaturation in muscle relative to what occurs in adipose tissue. Since all plant lipids contain large amounts of linoleic and linolenic acids and depot fats of sheep contain only trace amounts, ingested lipids must undergo a series of rather orderly alterations before ultimate deposition as triglycerides in adipocytes. The data presented in this paper suggest that the characteristic composition of sheep fat is dependent upon changes that occur in several different organs and tissues. The contributions of the gastro-intestinal tract appear to include hydrogenation of unsaturated fatty acids in the rumen, cecum and colon, the effect of bile secretions and rate of absorption of specific fatty acids. The liver probably exerts an influence by drawing upon the unsaturated fatty acid pool for phospholipid synthesis and cholesterol esterification. The final composition of depot lipids seems to be controlled by fatty acid synthesis and desaturation in muscle and adipose tissue.

LITERATURE CITED

Allen, E. A., A. R. Johnson, A. C. Fogerty, J. A. Pearson and F. S. Shenstone. 1967. Inhibition by

cyclopropene fatty acids of the desaturation of

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they are ultimately deposited in the adipose tissues. According to Edwards (1964) the fatty acid composition of lipids in alfalfa, milo and barley are about 75% C18 acids. This consists of approximately 5% stearic, 15% oleic, 25% linoleic and 30% linolenic acids. The highly unsaturated dietary plant lipids were probably hydrogenated in the rumen as stearic acid made up about 40 to 50% of the total fatty acids in the rumen lipids. Further evidence of rumen hydrogenation is noted in table 2. Almost 95% of the fatty acids in the radioactive trilinolein were at least partially hydrogenated and about 60% of the labeled linoleic acid was saturated to stearic acid. A shift in the 18 carbon acids occurred as the fatty acids proceeded from the rumen to the small intestines. The increases in the percentages of C18:1 and C18:2 acids (table 1) are probably a result of the contributions of fatty acids from the bile (Leat, 1965). The large increase in the radioactivity in C18:2 of the lipids from the sheep dosed with 14C-Trilinolein (table 2) in the small intestine, liver, and blood may indicate an uptake of linoleic acid by the liver for phospholipid formation. The circulating phospholipids are then probably partially deposited in muscle and adipose tissue and partially returned to the small intestine via the bile. The reduction in percentages of C18:1 and C18:2 in the lipids as they move through the small intestine to the colon (table 1) probably results not only from preferential absorption of unsaturated fatty acids as indicated by Miller and Cramer (1969) but also from further hydrogenation by cecal and colonic microorganisms. Ward et aL (1964) have presented evidence that hydrogenation occurs in the cecum and colon. Stearic acid was the predominant fatty acid in the liver lipids. This probably was the result of a more rapid removal of the C18 unsaturated acids from the liver in the phospholipid fraction coupled with the probability that ruminant liver does not contain desaturating enzymes (R. Reiser, personal communication). The fatty acid composition of subcutaneous adipose tissue and muscle lipids in table 1 compare favorably with those reported in the literature for mature sheep (Hartman and Shorland, 1961; Beare, 1962) in that the three major components, palmitate, stearate and oleate, make up about 31, 24 and 37%, respective-

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CRAMER AND MILLER Miller, L. G. 1967. Lipid digestion and metabolism in sheep. Ph.D. Thesis. Colorado State University, Fort Collins. Miller, L. G. and D. A. Cramer. 1969. Metabolism of naturally occurring and 14 C-labeled triglycerides in the sheep. J. Anim. Sci. 29:738. Raju, P. K. and R. Reiser. 1967. Inhibition of fatty acyl desaturase by cyclopropene fatty acids. J. Biol. Chem. 242: 379. Reiser, R. and H. G. R. Reddy. 1956. The hydrogenation of dietary unsaturated fatty acids by the ruminant. J. Amer. Oil Chem. Soc. 33:155. Schroepfer, G. J. and K. Bloch. 1965. The stereospecific conversion of stearic acid to oleic acid. J. Biol. Chem. 240: 54. Shorland, F. B., R. O. Weenink, A. T. Johns and I. R. C. McDonald. 1957. The effect of sheep-rumen contents on unsaturated fatty acids. Biochem. J. 67: 328. Thrall, B. E. and D. A. Cramer. 1971. The composition of some beef cattle lipids. Colo. State Univ. Exp. Sta. Tech. Bull. 111. Ward, P. F. V., T. W. Scott and R. M. C. Dawson. 1964. The hydrogenation of unsaturated fatty acids in the ovine digestive tract. Biochem. J. 92:60.

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stearic acid in hen liver. Lipids. 2:419. Beare, J. L. 1962. Composition of Fats. Fatty acid composition of food fats. J. Age. Food Chem. 10:120. Cramer, D. A. and J. A. Marchello. 1964. Seasonal and sex patterns in fat composition of growing lambs. J. Anim. Sci. 23:1002. Cramer, D. A., R. A. Barton, F. B. Shorland and Z. Czochanska. 1967. A comparison of the effects of white clover (Trifolium repens) and of perennial ryegrass (Lolium perenne) on fat composition and flavour of lamb. J. Agr. Sci. 69:367. Dole, V. P. and H. Meinertz. 1960. Microdetermination of longchain fatty acids in plasma and tissues. J. Biol. Chem. 235:2595. Edwards, H. M., Jr. 1964. Fatty acid composition of feedstuffs. Georgia Agr. Exp. Sta. Bull. N. S. 36. Hartman, L. and F. B. Shorland. 1961. Fatty acid composition and other characteristics of subcutaneous fat from New Zealand oxen and sheep. New Zealand J. Sci. 4:16. I.tat, W. M. F. 1965. Possible function of bile and pancreatic juice in fat absorption in the ruminant. Biochem. J. 94:21P. Mead, J. F. 1961. Synthesis and metabolism of polyunsaturated acids. Fed. Proe. 20:952.