POSITIONAL DISTRIBUTION OF FATTY ACIDS IN TIUACYLGLYCEROLS OF SEAL BLUBBER OIL

UDAYA N. WANASUNDARA and FEREJDOON SHAHIDI

Department of Biochemistry Memorial University of Newfoundland

St.John's, NF, AIB 3x9 Canada

Received for Publication August 22, 1996 Accepted for Publication October 18, 1996

ABSTRACT

The positional distribution of long-chain 0 3 polyunsaturated fatty acid namely eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) in the triacylglycerol (TAG) molecules of seal blubber oil was determined and compared with that of commercially available menhaden oil via stereospecijk analysis. In seal blubber oil, EPA, DPA and DHA occurred mainly in the sn-I andsn-3positions of the TAG molecules. The amounts were EPA, 8.36 and 11.2; DPA, 3.99 and 8.21; and DHA, 10.5 and 17.9%, respectively. In menhaden oil, DPA and DHA occurred mainly in the sn-2 position of the TAG at 3.1 I and I7.2%, respectively. However, EPA was equally distributed in the sn-2 and sn-3 positions and waspresent only in minute amounts in the sn-1 position.

INTRODUCTION

Long-chain 0 3 polyunsaturated fatty acids (03-PUFA), which are characteristic components of marine oils, have become a major focus of research because of their ability to prevent and possibly cure cardiovascular disease. The beneficial health effects of 03-PUFA have been ascribed to their ability to lower serum triacylglycerol (TAG) and cholesterol levels and enhance their excretion, to increase membrane fluidity and by being converted to eicosanoids, thus reducing thrombosis (Kinsella 1986). The 0 3 fatty acids are also considered essential for normal growth and development throughout the life cycle of humans (Kinsella 1986; Banden and Carroll 1986).

A significant amount of docosahexaenoic acid @HA) is found in human milk (Yongmanitchai and Ward 1989). High levels of DHA are also found in human brain and retina. Studies on nonhuman primates and human newborns indicate that DHA is essential for normal functional development of the retina and brain, particularly in premature infants. Therefore, DHA in breast-milk or infant formula

Journal of Food Lipids 4 (1997) 51-64. AN Rights Reserved. OCopyright 1997 by Food & Nutrition Press, Inc., Trumbull, CT 06611 51

52 U.N. WANASUNDARA and F. SHAHID1

is supposed to meet the requirements of developing human brain and visual parts (Neuringer ef ul. 1988). Because 0 3 fatty acids are essential for growth and development throughout the life cycle, they should be included in the diets of humans of all age groups. The a3 and 06 fatty acids are not interconvertible in the human body and are important components of practically all cell membranes. Whereas cellular proteins are genetically determined, the PUFA composition of cell membranes is, to a great extent, dependent on the dietary intake. Therefore, appropriate amounts of 0 3 and 06 fatty acids need to be considered in making dietary recommendations and these two classes of PUFA should be distinguished because they are metabolically and functionally distinct and have opposing physiological functions. The balance of the two groups of fatty acids is important for homeostasis and normal development (Simopoulos 1991).

Intestinal absorption of fatty acids has been reported to be dependent on their arrangement in the TAG molecules. Investigation of the absorption of fatty acids in a canine model suggested that the positional distribution of fatty acids within the TAG molecules (sn- 1, sn-2 and sn-3) might affect the metabolic fate of fatty acids (Jensen et al. 1994). Filer ef ul. (1969) and Tomarelli et uf. (1968) made a similar observation in human infants. On the other hand, different microbial lipases have been used to prepare 0 3 concentrates from marine lipids via a hydrolysis process. Some microbial lipases have both positional and acyl-chah specificities (Brockerhoff 1970). Therefore, knowledge of the fatty acid distribution in TAG molecules may provide useful information when selecting appropriate microbial lipases for hydrolysis of specific fatty acids in order to prepare 0 3 concentrates. Stereospecific analysis allows determination of the distribution of fatty acids over the three positions of the TAG molecules (Brockerhoff 1971). The analysis is based on the modification of TAG by Grignard degradation followed by synthesis of phosphatides and subsequent hydrolysis of fatty acids by stereospecific phospholipase A,.

Blubber (subcutaneous fat) constitutes approximately 25-30% of the seal carcass weight and is a rich source of 03-PUFA. "herefore, seal bhbber oil (SBO) may potentially be used in food and pharmaceutical applications. However, chemical charackristics of SBO have not been adequately studied. Therefore, over past few years in our laboratory we have intensified our research activities in order to characterize and utilize this important source of lipid. As part of our on going research we have determined the positional distribution of fatty acids in the TAG of SBO and compared it with that in the commercially available menhaden oil.

MATERIALS AND METHODS

Blubber of harp seal was obtained from local sources in Newfoundland. The blubber was then rendered, refined and bleached, as described elsewhere (Shahidi et ul. 1994). The refmed-bleached seal blubber oil (SBO) was deodorized in a

TRIACYLGLYCEROLS OF SEAL BLUBBER 53

laboratory-scale vacuum steam distillation apparatus. Refined, bleached and deodorized (RBD) menhaden oil (MHO) was obtained fiom Zapata Protein (USA) Inc. (Reedville, VA). Standard samples of fatty acid methyl esters (FAME) were purchased fiom either Supelco (Mississauga, ON) or Nu-Chek-Prep Inc. (Elysian, MN). All other chemicals used in this study were of ACS-grade or better quality.

Purification of Seal Blubber Oil (SBO) And Menhaden Oil (MHO)

Removal of nontriacylglycerols from SBO and MHO was carried out using column chromatography (1.25 cm internal diameter and 10 cm height) on silicic acid (100-200 mesh size, Mallinckrodt Canada Inc.). The column was first washed with hexane and then 1.25 g oil was introduced onto it. Hexane (50 mL) was added to the column followed by 10% (v/v) diethyl ether in hexane (250 mL) for elution. The solvent was then removed under vacuum at 40C using a rotary evaporator and recovered oil was passed though a layer of anhydrous sodium sulfate. In order to prevent oxidation of purified oils, a few crystals of butylated hydroxytoluene (BHT) were added to the mixture.

Reaction of Grignard Reagent With Purified Seal Blubber Oil (SBO) And Menhaden Oil (MHO)

Grignard reaction was performed on purified SBO and MHO according to the method described by Brockerhoff et ul. (1 963) and Brockerhoff (1 97 1) with some modifications. The purified oil (1 g) was dissolved in anhydrous diethyl ether (50 mL) and mixed with methyl magnesium bromide (3.5 mL, 3.0 M CH,MgBr in diethyl ether, Sigma Chemical Company, St. Louis, MO). The Grignard reaction was allowed to proceed with continuous stirring until a clear solution was obtained. To stop the reaction, glacial acetic acid (1 .O mL) was slowly added to the mixture followed by 10 mL of a 10% (w/v) boric acid solution in order to minimize acyl migration. Stirring of the reaction mixture was continued for another 2 to 3 min. The whole mixture was then transferred to a separatory hnnel and allowed to separate into two layers. The top ether layer was removed and the lower aqueous layer was washed twice with diethyl ether. The combined ether layers were washed successively with 10 mL of water, 10 mL of 2% (w/v) aqueous sodium bicarbonate and 10 mL of water and then dried over anhydrous sodium sulfate.

Separation of Individual Lipids after Grignard Reaction

Products of the Grignard reaction were dissolved in a minimum amount of chloroform and applied to several TLC plates (20 x 20 cm; Silica gel, 60 A mean pore diameter, 2-25 pm mean particle size, 500 pm thickness, with dichlorofluorescein, Sigma) impregnated with 5% (w/v) boric acid. The plates were developed in two different solvent systems of diethyl ether-petroleum ether

54 U.N. WANASUNDARA and F. SHAHIDI

FIG. 1. THIN LAYER CHROh4ATOGRAM OF GRIGNARD DEACYLATION PRODUCTS

(boiling point 30-60C) (8:92, v/v) and diethyl ether-petroleum ether (40:60, v/v), respectively. After drying, the bands were located by viewing under short (254 nm) and long (356 nm) UV lights (Spectraline, Model ENF-24OC, Spectronics Co. Westbury, NY). From the separated bands of triacylglycerol (TAG; R, = l.O), tertiary alcohol = 0.7), 1,2- (1,2-DAG) and 2,3-diaclylglycerols (2,3-DAG; R, = 0.3), 1,3-diacylglycerol(l,3-DAG; & = 0.4) and monoacylglycerol (MAG; & = 0.03) (Fig. l), the bands of 1,2- and 2,3-diacylglycerols were scraped and subsequently extracted with diethyl ether. The ether layer was evaporated under nitrogen to obtain 1,2- and 2,3-diacylglycerols. After removing a small sample for fatty acid analysis, the diacylglycerol fractions were used to prepare synthetic phospholipids.

Preparation of Synthetic Phospholipids from Diacylglycerol Fraction

The 1,2- and 2,3-diacylglycerols were dissolved in 1 .O mL of diethyl ether and mixed with 2.5 mL pyridine-diethyl ether-phenyl dichlorophosphate (1: 1:0.5, v/v/v). The reaction mixture was then allowed to stand at room temperatme for 1 .O h, after which 5 mL of pyridine, 3 .O mL of diethyl ether and few drops of water were added while cooling in an ice bath. The content of the flask was subsequently mixed with 86 mL of methanol-water-chloroform-triethylamine (30:25:30: 1, v/v/v/v). After standing, the lower chloroform layer containing the synthetic

TRIACYLGLYCEROLS OF SEAL BLUBBER 55

phospholipids (1,2-diacy1-3-phosphatide and 2,3-diacyl-l-phosphatide) was separated, and the solvent removed at 40C using a rotary evaporator. The recovered synthetic phospholipids were used for stereospecific hydrolysis by the phospholipase A, enzyme.

Stereospecific Hydrolysis of Synthetic Phospholipids by Phospholipase A,

The synthetic phospholipids (1,2-diacy1-3-phosphatide and 2,3-diacyl- 1 - phophatide) were dissolved in 3.0 mL of diethyl ether and transferred to a solution containing 15 mL of 0.1 M triethylammonium bicarbonate (PH 7.9, 100 pL of 0.1 M calcium chloride and 2.0 mg of phospholipase A, (EC. 3.1.1.4; Sigma) obtained from snake venom (Crotalus adamantus). The mixture was then shaken gently overnight in a Gyrotory water bath shaker (Model G76, New Brunswick Scientific Co. Inc., New Brunswick, NJ) and water in the mixture was evaporated at 40C using a rotary evaporator. In order to prevent foaming during evaporation, 15 mL of isobutanol was added to the mixture.

The hydrolyzed products were dissolved in 1 .O mL of chlorofodmethanol (1 : 1, v/v) containing one drop of glacial acetic acid. The dissolved hydrolytic products were applied to TLC plates impregnated with 5% (w/v) boric acid. The plates were developed in diethyl ether-petroleum ether (40:60, v/v), dried in a fumehood and then kept over concentrated aqueous ammonia for 10 min and redeveloped in aqueous ammonia-methanol-diethyl ether (2: 15:83, v/v/v). After drying, the bands were located by viewing under short (254 nm) and long (356 nm) W light. The separated bands of free fatty acids (hydrolyzed from sn-2 position of lY2-diacyl-3-phosphatide; &= 0.7 l), unhydrolyzed 2,3-diacyl-l-phosphatide (RF = 0.49), lysophosphatide (%= 0.05) and traces of 1,2- and 2,3-diacylglycerols & = 0.9 1) (Fig. 2) were scraped off and extracted into chloroform-methanol(1: 1, v/v). After removing a small portion of the sample for fatty acid analysis, the unhydrolyzed 2,3-diacyl- 1 -phosphatide fraction was hydrolyzed by porcine pancreatic lipase using the procedure described in the following section in order to obtain 2-monoacyl- 1-phosphatide and free fatty acids (hydrolyzed from sn-3 position). All separated lipid fractions were analyzed for their fatty acid composition by employing a gas chromatographic procedure.

Hydrolysis of Seal Blubber Oil (SBO) and Menhaden Oil (MHO) by Pancreatic Lipase

Hydrolysis of purified oils as well as separated 2,3-diacyl- I-phosphatide by pancreatic lipase was carried out according to the method described by Christie (1982). Tris-hydrochloric buffer (5 mL; 1.0 M, pH 8.0), 0.5 mL of calcium chloride (2.2%, w/v) and 1.25 mL of sodium taurocholate (0.05Y0, w/v) were added to 25 mg of the oil in a glass test tube. The whole mixture was allowed

56 U.N. WANASUNDARA and F. SHAHIDI

FIG. 2. THIN LAYER CHROMATOGRAM OF PRODUCTS OF PHOPHOLIPASE A2 HYDROLYSIS

to equilibrate at 40C in a water bath for 1.0 min and subsequently 5.0 mg of porcine pancreatic lipase (EC. 3.1.1.3; Sigma) were added to it. The mixture was then placed in a Gyrotory water bath shaker at 200 rpm under nitrogen for 8 to 10 min at 40C. Ethanol (5 mL) was added to stop the enzymatic hydrolysis followed by addition of 5.0 mL of 6.0 M HCl. The hydrolytic producls were extracted three times with 50 mL of diethyl ether and the ether layer was washed twice with distilled water and dried over anhydrous sodium sulfate. After removal of the solvent under vacuum at 30C, the hydrolytic products were separated on TLC plates impregnated with 5% (w/v) boric acid. The plates were developed using hexane-diethyl ether-acetic acid (70:30:1, v/v/v). After drying, the bands were located by viewing under short (254 nm) and long (356 nm) UV lights. The bands were scraped off and their lipids were extracted into chloroform-methanol (1: 1, v/v) or diethyl ether and subsequently used for fatty acid analysis by a gas chromatographic procedure.

Preparation of Fatty Acid Methyl Esters ( F A M E s )

Fatty acid composition of lipids was determined by their conversion to methyl esters. About 10 mg of each oil was weighed into a 6 mL well-cleaned Teflon-

TRIACYLGLYCEROLS OF SEAL BLUBBER 57

lined, screw capped conical vial. The internal standard (250 ng/100pL chloroform, methyl tricosanoate; C23:O) was added to the vial and the solvent in the oil-internal standard mixture was evaporated under a stream of nitrogen. Transmethylation reagent (2 mL, freshly prepared 6 mL of concentrated sulfuric acid made up to 100 mL with spectral grade methanol and 15 mg of hydroquinone as an antioxidant) was added to the sample vial and mixed by vortexing. The mixture was incubated overnight at 60C and subsequently cooled (Wanasundara and Shahidi 1996). Distilled water (1 .O mL) was added to the mixture and after thorough mixing, extracted three times with 1.5 mL of pesticide-grade hexane. A few crystals of hydroquinone were added to each vial prior to extraction with hexane. Hexane layers were separated, combined and transferred to a clean tube and then washed two times with 1.5 mL of distilled water. In the frst wash, the aqueous layer was removed and in the second wash, the hexane layer was separated and evaporated under a stream of nitrogen. Fatty acid methyl esters were then dissolved in 1 .O mL of carbon disulfide and used for gas chromatographic analysis (see below).

Analysis of Fatty Acid Methyl Esters (FAMEs) by Gas Chromatography (GC)

A Hewlett Packard 5890 Series I1 gas chromatograph (Hewlett Packard, Toronto, ON) equipped with an Hp-INNOWAX column (0.25 mm diameter, 30 m length, 0.25 pm film thickness; Hewlett Packard, Toronto, ON) was used for analyzing FAMEs. The oven temperature was initially 220C for 10.25 min and then ramped to 240C at 30C/min and then held there for 9.0 min. The injector and detector (flame ionization, FID) temperatures were both at 270C. Ultra high pure (UHP) helium was used as a carrier gas (15mL/min). HP 3365 Series I1 ChemStation software (Hewlett Packard, USA) was used for data handling. The FAMEs were tentatively identified by comparison of their retention times with those of authentic standard mixtures. The area under each peak was calculated on a weight percentage basis using methyl tricosanoate (C23:O) as an internal standard.

RESULTS AND DISCUSSION

Stereospecific analyses of SBO and MHO and the intermediates involved are shown in Fig. 3. In the first step, TAG of both SBO and MHO were hydrolyzed by porcine pancreatic lipase in order to split fatty acids in the sn-1 and sn-3 positions. The resultant 2-monoacylglycerols, accurately provide the fatty acid composition of the sn-2 position of TAG. In the second step, TAG of both SBO and MHO were modified by Grignard degradation using CH,MgBr. Laakso and Christie (1990) and Itobashi et al. (1990) used Grignard reagent to obtain partially deacylated triacylglycerols, i.e., diacylglycerols (Fig. 4). The products of the

58 U.N. WANASUNDARA and F. SHAHID1

TAGS

rl Pancreatic

2 4 + Freefattyadds

L-3 A

LOH B

FIG. 3. PROCEDURE FOR THE STEREOSPECIFIC ANALYSIS OF TRIACYLGLYCEROL (TAG) OF SEAL BLUBBER OIL (SBO) AND MENHADEN OIL 0

TRIACYLGLYCEROLS OF SEAL BLUBBER 59

0 - C - R R'-C-0 7 $*& 0

- + 0 MgBr 0 ' { fl + C%-C-R II

6- s+ 9 - C - R + q M g B r &W R'-c-0

Ketone Ketone

J 0-C-R"

IH+ 0 T O H

R-c-0 'I 7

0-c-R"

1,Zdiacylglycerol and/or 2,3diacyIglycerol

.: + :O: MgBr I I

q - C - R

cH3 iH+ OH

and/or I

13-diacylglycerol and/or monoacylglycerol I C% -C-R

k 3 Tertiary alcohol

FIG. 4. CHEMICAL REACTIONS INVOLVED DURING GRIGNARD DEGRADATION OF TRIACYLGLYCEROL (TAG) OF SEAL BLUBBER OIL (SBO) AND MENHADEN OIL (MHO)

60 U.N. WANASUNDARA and F. SHAHIDI

reaction are a ketone and acylglycerols. The resulting ketone reacts further with the Grignard reagent to yield a tertiary alcohol. The products of the Grignard degradation of TAG which were separated on TLC plates (Fig. 1) exhibited a band with an Rf of 0.30 (1,2- and 2,3-diacylglycerols). The compounds of this band were isolated and used to prepare synthetic racemic phosphatides by reacting them with dichlorophenylphosphate. This reaction produced 1,2-diacylglycer0-3- phosphatide (L-isomer) and 2,3-diacylglycero- 1-phosphatide @-isomer). These phosphatides were hydrolyzed by stereospecific phospholipase A, enzyme extracted from snake venom. This enzyme reacts only with 1,2-diacylglycero-3- phosphatide (L-isomer; ~ t ~ r a l l y present) and releases h e fatty acids from the sn-2 position (Verheij and Dijkstra 1994). The products of phospholipase A, hydrolysis (free fatty acids from sn-2 position and L-lysophophatide) and the unchanged 2,3- diacylglycero-1-phosphatide @-isomer; un-natural) were sepatated on a TLC plate (Fig. 2). The band with an Rf value of 0.05 (L-lysophosphatide) was isolated, extracted into chloroform-methanol (1 : 1 , v/v) and then used for fatty acid analysis. These allowed identification of the fatty acid composition in the sn- 1 position of TAG of SBO and MHO.

Fatty acid distribution in the three different positions of TAG of SBO and MHO are given in Tables 1 and 2, respectively. In SBO, saturated fatty acids such as 14:0, 15:0, 16:O and 17:O were preferentially located in the sn-2 position, followed by the sn-1 and sn-3 positions. However, the saturated fatty acids in MHO were randomly distributed over the three positions. SBO contained very high amounts (over 59%) of monounsaturated fatty acids, in which 18:l was preferentially located in the sn-1 position, whereas 16: 1 and 20: 1 were abundant in the sn-2 and sn-3 positions, respectively. In MHO, monounsaturated fa#y acids were randomly distributed. SBO and MHO were also different in the dominancy and distribution of long-chain 0 3 fatty acids in their TAG molecules. In SBO, eicosapentaenoic acid (EPA; 20:503), docosapentaenoic acid @PA; 22503) and DHA (22:603) occurred mainly in the sn-1 and sn-3 positions of TAG and their quantities were EPA, 8.36 and 11.2%; DPA, 3.99 and 8.21%; and DHA, 10.5 and 17.9%, respectively. In MHO, DPA and DHA occurred mainly in the sn-2 position of TAG at 3.1 1 and I7.2%, respectively. However, EPA was equally distributed in the sn-2 (17.5%) andsn-3 (16.3%) positions and was present only in small amounts (3.12%) in the sn- 1 position.

Brockerhoff et ul. (1966) have pointed out the general tendency of long-chain PUFA to be preferentially esterified in the sn-2 position of fish oil TAG, whereas in marine mammal lipids, these fatty acids are mainly located in the sn-1 and sn-3 positions. Recently, Aursand et ul. (1995) determined the positional distribution of 0 3 fatty acids in cod liver and Atlantic salmon oils (fish) and seal blubber oil (marine mammal) by high-resolution "C nuclear magnetic resonance spectroscopy and found that in cod liver and salmon oils DHA was concentrated in the sn-2 position of TAG, whereas EPA was nearly randomly distributed in all three

TRIACYLGLYCEROLS OF SEAL BLUBBER 61

positions. In seal oil, DHA was primarily present in the sn-1 and sn-3 positions. Similar observations were made by Brockerhoff et al. (1963; 1966) for fish (herring and mackerel) and marine mammal depot lipids (seal blubber oil). However, the major drawback of their method is that their results for the sn-3 position is subject to cumulative error because fatty acid distribution in this position was not determined directly. Ando et al. (1992) have determined the positional distribution of fatty acids in TAG of fish oils (capelin, herring, menhaden, sardine and saury) by high-performance liquid chromatography using

TABLE 1. FA'ITY ACID DISTRIBUTION IN DIFFERENT POSITIONS OF TRIACYLGLYCEROLS OF

SEAL BLUBBER OIL (SBO)

Position Fatty acids SBO

(w/w %) Triacylglycerol sn-I" ~ n - 2 ~ 5n-3'

14:O 14:109 15:O 16:O 16:107 17:O 17:l 18:O 18:l' 18:206 18:3w3 18:403 20:o 20: 109 20:206 20:3' 20:406 20:503 22:o 22:1011 22:2 22:406 22:503

3.73 * 0.08 1 .09 f 0.04 0.23 f 0.00 5.98 f 0.03 18.0 f 0.04 0.92 f 0.00 0.55 f 0.02 0.88 f 0.00 26.0 f 0.06 1.51 f 0.02 0.59 f 0.00 1 .oo f 0.02 0.1 1 f 0.00 12.2 f 0.02 0.16 f 0.00 0.14 f 0.00 0.46 f 0.01 6.41 f 0.08

2.01 f 0.04

0.11 f 0.01 4.66 f 0.01

2.23 f 0.12 0.23 f 0.00 0.10 f 0.00 3.12 f 0.13 8.22 f 0.16 0.79 f 0.01 0.20 f 0.00 1.01 f 0.01 38.3 f 1.00 0.32 f 0.00 0.96 f 0.01 1.82 f 0.16 0.1Of 0.00 13.0f0.10 0.21 f 0.00 0.19 f 0.00 0.21 f 0.02 8.36 f 0.12

3.00 f 0.04

0.01 f 0.00 3.99 f 0.05

8.08 * 0.01 2.35 f 0.03 1.25 f 0.01 12.4rt0.15 35.3 f 0.80 1.80 f 0.01 1.30 f 0.00 1.64 f 0.06 22.6 f 1.00 1.24 f 0.09 0.45 f 0.01 0.45 f 0.02 0.43 f 0.00 4.33 f 0.13 0.15 f 0.00 0.13 f 0.00 0.14 f 0.00 1.60 f 0.09

0.13 f 0.01

0.05 f 0.00 0.79 f 0.06

0.12 * 0.00 0.22 f 0.00 0.41 0.01 2.99 f 0.04 12.3 f 0.12 0.80 f 0.01 0.12 f 0.00 0.71 f 0.02 18.OfO.17 2.01 f 0.08 1.21 f 0.01 0.33 f 0.09 0.09 f 0.00 17.2+O0.ll 0.20 f 0.00 0.22 f 0.01 0.91 f 0.01 11.2 f 0.04

3.25 * 0.03

0.22 f 0.00 8.21 f 0.08

22:603 7.58 f 0.02 10.5 f 0.10 2.27 f 0.12 17.9f0.11

"L-lysophosphatide (F'), b2-Monoacylglycerol (BI), '3-Fatty acids (HI) 'Refer to Fig. 3, 'Includes all isomers

62 U.N. WANASUNDARA and F. SHAHIDI

TABLE 2. FATTY ACID DISTRIBUTION IN DIFFERENT POSITIONS OF TRIACYLGLYCEROLS

OF h4ENHADEN OIL (MHO)

Position Fatty acids MHO (wh %) Triacylglycerol sn-1' sn-2b sn-3"

14:O 14:lw9 15:O 16:O 16:107 17:O 17:l 18:O 18:l' 18:2w6 18:303 18:403 20:o 20: 109 20:206 20:3' 20:406 20:5w3 22:o 22:1011 22:2 22:406 22:503 22603

8.32 f 0.12 0.38 f 0.01 0.71 f 0.02 17.1 f 0.24 11.4 f 0.13 2.45 f 0.12 1.86 f 0.03 3.33 f 0.02 12.1 f 0.12 1.42 f 0.09 1.82 f 0.00 2.90 f 0.05 0.20 f 0.01 1.44 f 0.06 0.21 f 0.00 0.46 f 0.01 0.83 f 0.02 13.2f0.18 0.12 f 0.00 0.12 f 0.05 0.02 f 0.00 0.19 f 0.03 2.40 f 0.03

12.1 f 0.10 0.30 f 0.01 1.02 f 0.01 24.1 f 0.55 18.1 f 0.32 4.39 f 0.12 2.11f0.09 1.21 f 0.02 13.1 f 0.21 0.81 f 0.00 1.12 f 0.01 4.51 f 0.02 0.21 f 0.00 0.71 f 0.00 0.40 f 0.00 0.59 f 0.00 1.11 f 0.01 3.12 f 0.03 0.21 f 0.02 0.13 f 0.00 0.20 f 0.00 0.20 f 0.01 1.21 f 0.01

10.5 f 0.09 0.60 f 0.00 0.3 1 f 0.00 19.9f 0.13 10.6 f 0.18 3.65 f 0.03 2.23 f 0.13 2.06 f 0.00 7.47 f 0.01 0.49 f 0.00 0.41 f 0.01 0.34 f 0.03 0.1 1 f 0.00 0.50 f 0.01 0.14 f 0.00 0.42 f 0.00 1.13 f 0.02 17.5 f 0.19 0.08 f 0.00 0.12 *0.00 0.15 f 0.00 0.31 f 0.02 3.11 f0.02

5.23 f 0.01 0.16 f 0.00 0.80 f 0.01 6.91 f 0.01 9.20 f 0.10 1.01 f 0.00 1.23 f 0.03 4.12 f 0.01 13.6 f 0.09 3.11*0.03 0.99 f 0.03 4.94 f 0.01 1.21 f 0.01 2.20 f 0.03 0.1Of 0.00 0.61 f 0.00 0.27 f 0.01 16.3 f 0.18 0.21 f 0.02 0.09 f 0.00 0.18 f 0.00 0.04 f 0.00 2.31 f 0.05

10.1 320.11 4.1 1 f 0.03 17.2 f 0.19 6.12 f 0.02

"L-lysophosphatide PI), ,%Monoacylglycerol @I), c3-Fatty acids (HI) 'Refer to Fig. 3, 'Includes all isomers

a chiral stationary phase and found that in these oils DHA was present mainly in the sn-2 position of TAG. However, Ota et ul. (1994) found that in flounder liver and flesh lipids there was no preference for the sn-2 position in contrast to the general tendency for distribution of long-chain PUFA of fish oils in this position.

CONCLUSIONS

The positional distribution of fatty acids in the TAG molecules of SBO and MHO was determined by employing stereospecific analysis; EPA, DPA and DHA

TRIACYLGLYCEROLS OF SEAL BLUBBER 63

were located mainly in the sn-1 and sn-3 positions of the TAG molecules of SBO. In MHO, DPA and DHA were found mainly in the sn-2 position of the TAG, however, EPA was equally distributed in the sn-2 and sn-3 positions and was present only in small amounts in the sn-1 position. Therefore, EPA, DPA and DHA in SBO might be assimilated in the body more effectively than those in MHO (fish oil). During digestion, fatty acids in the sn-1 and sn-3 positions of the TAG are liberated by a position specific enzyme such as pancreatic lipase, but the fatty acids attached to the sn-2 position of the TAG are absorbed and distributed in the body in the chylomicron form. However, clinical studies should be carried out to verify this latter assumption.

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