Positional Analyses of Triacylglycerol Fatty Acids in the Milk Fat of the Antarctic Fur Seal (Arctocephalus gazella) Donald L. Pupplone a,*, Cindy M. Kuehlthau b, Ronald J. Jandacek b and Daniel P. Costa a along Marine Laboratory, Institute of Marine Science, University of California, Santa Cruz, California 95064 arid b'vhe Procter and Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45239

637

The positional distribution of fatty acids has been deter- mined for the milk triacylglycerols of the Antarctic fur seal, Arctocephalus gazella. Of particular interest was the positional distribution of the polyunsaturated n-3 fatty acids in milk triacylglycerols (TG). In adipocytes of pinnipeds, TG are synthesized with the n-3 fatty acids primarily in the sn-l,3 positions. To determine the posi- tional distribution, extracts of enzymatically digested iipids were separated by thin-layer chromatography, and the constituent fatty acids were separated and quantified by gas-liquid chromatography. Monoenoic and saturated fatty acids comprised over 75% of the total, the ratio of monoenoic to saturated fatty acids being 2:1. The percent content of the long-chain n-3 fatty acids, 20:5, 22:5 and 22:6, ranged between 15-20%. The positional analyses revealed that at the sn-2 position of milk TG, saturated fatty acids were in excess (57%), and the content of n-3 fatty acids was less than 5%. More than 80% of the n-3 fatty acids in milk were located in the sn-l,3 positions. The data indicate that in pinnipeds TG are synthesized in the mammary gland and adipose tissue with fatty acids having similar positional distributions. Lipids 27, 637-639 {1992).

As a group, pinnipeds produce milk with an exception- ally high fat content (1-10), ranging from 19% in the Galapagos sea lion (1) to 61%, the highest value reported for any mammal, in the hooded seal (21. In the milk of the various species, 18:1 (17-47%), 16:0 {9-22%) and 16:1 (6-19%) 16-8,11-17) consistently were found to be the predominant three fatty acids. The polyenoic n-3 (20:5, 22:5 and 22:6) and the monoenoic (20:1 and 22:1) fatty acids common to the marine food web comprised between 15-40% of the total fatty acids in the milk of pinnipeds 16 - 8,11--13,16,17). Rather th an being synthesized de n ova the polyenoic and most likely the long-chain monoenoic fatty acids are derived from the diet of the lactating female

Brockerhoff and co-workers (18-20) showed that the fatty acid positional distribution in stort<[ triacylglycemls (TG) were different in pinnipeds than in cold blooded organisms. Therefore, we were interested in determining whether the mammary gland of pinnipeds adaptively ar- ranged the fatty acids in the milk TG to mimick the posi- tional distribution in the stored fat of prey animals. To answer this question and to gain a better understanding of the utilization and processing of lipids in these animals, the positional distribution was determined for the fatty acids in the milk of the Antarctic fur seal (Artocephalus gazella). Our data on the milk TG indicate that there is no such adaptation. Overall, the positional distribution

*To whom correspondence should be address~i at Department of Chemistry and B~ochemisr, ry, UCLA, 405 Hilgard Avenue, Los Angeles, CA 90024.

Abbreviations: TG, triacylglycerol; TLC, thin-layer chromatography.

of the fatty acids is very similar to data reported previ- ously for seal blubber (18-20}.

METHODS Milk samples (30-120 mL) were collected on Bird Island, South Georgia (54, 00"S, 38, 02'W) from four fur seals marked on the date of parturition. At the time of collec- tion, the animals, which could be approached to within 1-2 m, were captured with a rope noose attached to a 3-m pole Captured animals were then restrained using a specially constructed board as described by Gentry and Holt (21). Once restrained, females were weighed and then injected with 40 units of Oxytocin. After a few minutes, milk was manually expressed from the teat into plastic vials. Samples were kept frozen at -20~ until lipid analyses were carried out.

The milk lipids were extracted with chloroform/metha- nol (2:1, vol]vol). Extracts were weighed to obtain the per- cent lipid in the milk and then analyzed by tldn-layer chro- matography (TLC). The total fatty acids in the lipid ex- tracts were methylated with boron trifluoride after under- going saponification (22}. The distribution of fatty acid methyl esters was determined by gas-liquid chromatog- raphy. Methyl esters were separated on a Durabond 225 column (0.00025 • 30 m,.J&W Scientific, Folsom, CA) in a Hewlett-Packard (Kennet Square, PA) model 5890a equipped with a hydrogen flame detector. Column temper- ature was 220~ The carrier gas was helium and the flow rate was 1.42 mL/mm. The detector and injection port temperatures were 350~ The resulting peak areas were measured electronically with a Hewlett-Packard 3392a and normalized to give values as weight percent of total fatty acid.

To determine the positional fatty acid distribution, the lipid extracts were first hydrolyzed with porcine pan- creatic lipase (steapsin) in a 1.0 M Tris buffer (pH 8.0) as described by Mattson and Volpenhein (23). Following ex- traction, the enzymatic digestion products, 2-monoacyl- glycerols and free fatty acid released from the 1- and 3-positions, were separated by TLC. Using fatty acid and monogiycerel standards, both the digestion products and completion of the reaction could be monitored- The mono- glycerols and free fatty acids were scraped from the plate and recovered after a subsequent TLC separation with only the standards being visualized with iodine vapor. Methylation of fatty acids and analyses of the resulting methyl esters for each of the recovered fractions were done as described above

RESULTS

Analyses by TLC confirmed that the extracted lipid from fur seal milk consisted almost exclusively of TG. Although milk samples were obtained at different stages of the lac- tation cycle and the overall fat content in the milk varied from 30-52% (Table 1), individual variations in the per- cent content of the major fatty acids were quite small

LIPIDS, Vol. 27, no. 8 (1992)

638

D.L. PUPPIONE ET AL.

TABLE 1

Percent Fat Content in the Milk of Four Antarctic Fur Seals

Female (no.) Days postpartum Percent fat

247 0 52.3% 263 0 42.6% 257 17 29.7% 235 32 39.5%

TABLE 2

A Comparison of the Fat ty Acid Distributions in the Milk Triacylglycerols (TG} a of Four Antarctic Fur Seals and in the TG of Their Major Prey, E. superba

Antarctic fur seals (no.) E. superbab

Fatty 247 263 257 235 (weight acid Weight percent percent)

14:0 3.7 4.7 6.4 6.1 14.6 16:0 18.4 18.6 18.4 17.9 23.0 16:1 9.3 9.8 10.6 10.2 9.0 17:1 1.0 1.0 1.1 1.0 0.5 18:0 1.7 1.8 2.0 2.0 1.1 18:1 36.0 32.2 36.6 35.5 20.0 18:2 1.7 1.7 1.7 1.5 0.2 18:3 0.5 0.5 0.7 0.4 0.3 20:1 4.9 4.1 4.5 2.7 0.8 20:4 0.7 0.7 0.4 0.5 0.5 20:5 7.1 8.7 6.9 12.1 13.7 22:1 1.3 1.3 0.9 0.6 -- 22:5 2.0 2.1 2.2 2.4 0.2 22:6 7.1 8.0 6.0 5.5 8.0

Total 95.4 95.2 98.4 98.4 91.9

a Fatty acid distributions were obtained from a single analysis of each individual milk sample.

bReference 24.

(values are listed in Table 2). The fa t ty acids with less than 20 carbons comprised over 70% of the total. The remain- ing port ion consisted pr imari ly of 20:5 and 22:6, with the ratio of 20:5 to 22:6 vary ing from 1:2.2. The percentage of any one of the polyenoic fa t ty acids in Group 1 never exceeded 1.7%.

When the distr ibution a t the sn-2 position was exam- ined, a definite pa t t e rn could be discerned. Over 85% of these f a t ty acids were in order of relative amounts 16:0, 18:1, 16:1 and 14:0. From the da ta in Table 3, it can be calculated t ha t 73% of 16:0 and 61% of 14:0 in milk TG were located at the sn-2 position. The corresponding values for 16:1 and 18:1 were 43% and 17%, respectively. A random ar rangement would correspond to a value of 33% at the sn-2 position. In contrast, the percent content at the sn-2 position for any of the polyenoic f a t t y acids of Group 2 was low. At the sn4,3 positions, 20% of the f a t ty acids in the TG of fur seal milk were 20:5, 22:5 and 22:6.

DISCUSSION

The high fat content in pinniped milk is related to their amphibious existence, which separates lactat ion on shore from feeding a t sea (9,10). In the family Otariida~

TABLE 3

Positional Distribution of Fat ty Acids in Fur Seal Milk a

sn-2 Position sn-l,3 Positions

Saturated fatty acids 14:0 11.4 (2:4.3) 3.6 {-+1.5) 16:0 44.9 (_+1.8) 8.4 (_0.5) 18:0 0.5 (__-0.05) 2.6 (_+0.2)

Monoenoic fatty acids 16:1 13.8 (__-0.9) 9.2 (___1.2) 18:1 17.5 (__-4.0) 41.5 (+__3.9) 20:1 0.2 (+_0.21) 5.7 (+l.0) 22:1 1.5 (-+0.4)

Polyenoic fatty acids Group 2 b 18:2 2.2 (+0.3) 1.4 (-+0.08) 18:3 0.6 (+---0.17) 0.7 (-+0.08) 20:4 0.3 (-----0.19) 0.5 (_+0.15)

Polyeneic fatty acids Group 2 c 20:5 2.2 (-+0.4) 9.7 (__.2.5) 22:5 0.2 (__.0.26) 2.8 (-+0.2) 22:6 1.6 (-+0.8) 7.5 (_+1.3)

Totals 95.4 95.1

a Percent content values are means obtained from the analyses of four individual samples of fur seal milk. Standard deviations are given in parentheses.

bpolyenoic fatty acids Group 1 consists of polyunsaturated fatty acids commonly found in the diet of terrestrial animals.

c Polyenoic fatty acids Group 2 consists of polyunsaturated fatty acids commonly found in the diet of marine animals.

consis t ing of fur seals and sea lions, lactat ion on shore is interspersed between feeding trips to s ea These periods of foraging typical ly last between 1 and 7 d and then are followed by a suckling period of 1 to 3 d on shore {10). In the case of the Antarct ic fur seal, while a t sea the female will derive 52% of her calories from the fat s tored in krill {25). Such a diet enables the seal to provide her pup with milk enriched in lipids (26).

Our current s tudy is the first to report on the par t ia l posit ional distr ibution of the n-3 f a t ty acid in the milk of a pirmiped. The polyenoic n-3, as well as the monoenoic C20 and C~2 fa t ty acids, also common to the marine food web, were located pr imari ly in the sn-l,3 positions. The sn-2 position contained a high percentage of 16:0 (45%). Overall, the distr ibution was essentially the same as reported previously for the TG of seal blubber (18-20). In seals, the sn-2 position was found to contain most ly sa tura ted f a t ty acids, 14:0 and 16:0, with lesser amounts of monoenoic acids, 16:1 and 18:1. The n-3 fa t ty acids com- prised only a small percentage of the f a t ty acids at this position and were located primarily at the sn-l,3 positions. On the other hand, in fish and invertebrate TG, the n-3 fa t ty acids were located primarily at the sn-2 position, and 14:0 and 16:0 were the two major f a t ty acids a t the sn-l,3 posi t ions {18,19). There was no adapta t ion in the mam- mary gland to provide suckling pups with die tary fa ts wi th positional dis tr ibut ions mimicking those found in future prey animals in the ocean.

Prior to our study, the f a t ty acids in the milk TG had been reported for three other species of otari ids or

LIPIDS, Vol. 27, no. 8 (1992)

FUR SEAL MILK TRIACYLGLYCEROL FATTY ACIDS

639

eared seals, the Nor thern fur seal (Callorhinus ursinus) (8), the California sea lion (Zalophus californianus) (16) and the Galapagos fur seal (Arctocephalus galapagoensis) (17). Many more studies have been done on phocids or earless seals (3,6,7,11-16). Our da ta on the total f a t ty acid com- position are in general agreement with these other studies which show the milk fa t to be enriched, par t icular ly in monoenoic and sa tura ted f a t ty acids (7,8,11-17). In mos t studies, including our own, the ratio of monoenoic to sa tura ted f a t ty acid was approximate ly two. Except ions to this t rend were da ta obtained on the milk of the harp seal (Pagophilus groenlandicus), hooded seal (Cystophora cristata) and the California sea lion. In the seal studies (3,6), the milk had a low content of sa tura ted f a t t y acids result ing in the ratio having a value greater than 3.8. However, values reported by Cook and Baker (13) for harp seal milk agree with data obtained in other species. In the s tudy of the California sea lion milk, the relative m o u n t s of sa tu ra ted and monoenoic acids were a lmost equal and the result ing ratio was sl ightly greater than one (16). Observed differences in the fa t ty acid composition of milk TG may be related to variat ions in the composit ion of the die tary fat of these animals. The long chain polyenoic acids are a t comparable levels in bo th the milk and the principal prey, E u p h a u s i a superba (24), of the Antarct ic fur seal (see Table 2).

In addition to obtaining da ta on the major energy source of suckling fur seals, our s tudy may provide in- s ights into ways to supplement infant formula diets with n-3 fa t ty acids. There is growing evidence t ha t the n-3 fa t ty acids are essential in the neonatal period in humans (27,28). Studies have been done with formulas supple- mented with fish oils as a source of 20:5 and 22:6 n-3 fa t ty acids (28). We current ly are extending our studies of neonatal pinnipeds to determine whether variations in the location of n-3 f a t ty acids in dietary marine fa ts may be important . By s tudy ing animals before and after wean- ing, when they are switched to a diet of fish, we can deter- mine the effects on bo th p lasma membranes and lipopro- teins, in par t icular on the size and number of chylomicra. Hav ing natural diets with fats differing in the posit ional distribution of n-3 fa t ty acids, neonatal pinnipeds provide us with a unique animal model for addressing some of these issues.

ACKNOWLEDGMENTS D. P. Costa was supported by Division of Polar Programs National Science Foundation Grants 8311799 and 8618820. Logistic support generously was provided by the British Antarctic Survey, Cambridge, England. J. Mastro assisted in the collection of milk samples in the field. Many thanks go to J. P. Croxall of the British Antarctic Survey for making our studies at Bird Island possible

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[Received July 13, 1991; Revision accepted June 6, 1992]

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