Isovaleroyl triglycerides from the blubber and melon oils of the beluga whale (Delphinapterus...

8
Isovaleroyl Triglycerides From the Blubber and Melon Oils of the Beluga Whale (Delphinapterus leucas) CARTER LITCHFI ELD 1, Department of Biochemistry and Biophysics, Texas Agricu ltu ral Experiment Station, College Station, Texas 77843, and R.G. ACKMAN, J.C. SIPOS and C.A. EATON, Halifax Laboratory, Fisheries Research Board of Canada, Halifax, Novia Scotia ABSTRACT The fatty acid compositions of the blubber and melon oils from the beluga whale (Delphinapterus teucas) have been determined by gas liquid chromatography (GLC). The melon oil contains a high level (60.1 mole %) of isovaleric acid, substantial amounts of long chain branch- ed acids (16.9%), and very little polyun- saturated material (0.5%). The blubber oil contains less isovaleric (13.2%), fewer long chain branched acids (2.7%), and appreciable amounts (10.9%) of the poly- unsaturated acids typical of marine oils. The blubber and melon oils were also examined for lipid class composition by thin layer chromatography on silicic acid, direct GLC of the hydrogenated oil, and gel permeation chromatography. Both oils are composed almost entirely of triglycerides, which can be separated chromatographically into molecules con- taining 0, 1 and 2 isovaleric acid moieties. No triisovalerin could be detected. The blubber oil contains 68.9 mole % normal triacyl-, 24.2% diacyl-monoisovaleroyl-, and 7.0% monoacyl-diisovaleroyl-triglyc- erides (acyl = long chain acid). Mono- acyl-diisovalerin constitutes 86.7 mole % of the melon oil. This unusual compound may play a role in the echolocation system of the beluga whale. INTRODUCTION Isovaleric acid has been reported in the melon (forehead), jaw and blubber oils of dolphins (I-4), porpoises (5-9), the pilot whale (10), and the beluga whale (11). Apparently only this small group of marine mammals out of the entire animal kingdom is able to biosynthesize fatty oils containing this unusual fatty acid. Recent biological studies by Norris (12,13) indicate that the fatty tissues in the melon and jaw of dolphins, porpoises and toothed whales Ipresent address: Department of Biochemistry, Nelson Biological Laboratories, Rutgers-The State University, New Brunswick, New Jersey 08903. (i.e., the suborder Odontoceti) may play a major role in the echolocation system of these animals. It seems appropriate, therefore, to conduct a detailed study of Odontoceti head oils to determine if their unusual chemical structure might possibly be related to their role in echolocation. Lovern (7) and Varanasi and Malins (6) have reported that isovaleric acid occurs in mixed acid triglycerides of the melon, jaw and body oils from the porpoise Phocoena phocoena. On the other hand, Tsujimoto and Koyanagi (10) found both cetyl isovalerate and mixed isoval- eroylqong chain triglycerides in the head oil of the pilot whale Globicephala macrorhyncha. Varanasi and Matins (5) have also identified isovalerate wax esters in Tursiops gilli jaw oil. This paper reports a detailed comparison of molecular species of triglycerides found in the blubber and melon oils of the Arctic beluga whale, Delphinapterus leucas. EXPERIMENTAL PROCEDURES Materials Beluga melon oil (Delphinapterus leucas) was supplied by Churchill Whale Products Ltd., Churchill, Manitoba. It came from the com- bined melons of several whales caught in Hudson Bay in August 1966. Beluga blubber oil was obtained from commercial production by the same company. Gas Liquid Chromatography of Fatty Acids Triglycerides were converted to methyl esters by heating in a screw-cap (Teflon fined) centri- fuge tube with 5 ml 7% (w/v) BF3/CH3OH reagent per 100 mg as described by Morrison and Smith (14). Hexane rather than benzene was added to promote sample solubilization, since benzene was found to produce an artifact with a retention time on butanediolsuccinate polyester (BDS) columns very similar to that of methyl isobutyrate. Esters were normally con- centrated into the hexane layer for gas liquid chromatography (GLC) analysis by the addition of 1.5 ml water to the reaction tube. Occasional direct GLC study of homogeneous reaction mixtures was attempted to check the quantita- 674

Transcript of Isovaleroyl triglycerides from the blubber and melon oils of the beluga whale (Delphinapterus...

Page 1: Isovaleroyl triglycerides from the blubber and melon oils of the beluga whale (Delphinapterus leucas)

Isovaleroyl Triglycerides From the Blubber and Melon Oils of the Beluga Whale (Delphinapterus leucas) CARTER LITCHFI ELD 1, Department of Biochemistry and Biophysics, Texas Agricu ltu ral Experiment Station, College Station, Texas 77843, and R.G. ACKMAN, J.C. SIPOS and C.A. EATON, Halifax Laboratory, Fisheries Research Board of Canada, Halifax, Novia Scotia

ABSTRACT

The fat ty acid compositions of the blubber and melon oils from the beluga whale (Delphinapterus teucas) have been determined by gas liquid chromatography (GLC). The melon oil contains a high level (60.1 mole %) of isovaleric acid, substantial amounts of long chain branch- ed acids (16.9%), and very little polyun- saturated material (0.5%). The blubber oil contains less isovaleric (13.2%), fewer long chain branched acids (2.7%), and appreciable amounts (10.9%) of the poly- unsaturated acids typical of marine oils. The blubber and melon oils were also examined for lipid class composit ion by thin layer chromatography on silicic acid, direct GLC of the hydrogenated oil, and gel permeation chromatography. Both oils are composed almost entirely of triglycerides, which can be separated chromatographically into molecules con- taining 0, 1 and 2 isovaleric acid moieties. No triisovalerin could be detected. The blubber oil contains 68.9 mole % normal triacyl-, 24.2% diacyl-monoisovaleroyl-, and 7.0% monoacyl-diisovaleroyl-triglyc- erides (acyl = long chain acid). Mono- acyl-diisovalerin constitutes 86.7 mole % of the melon oil. This unusual compound may play a role in the echolocation system of the beluga whale.

INTRODUCTION

Isovaleric acid has been reported in the melon (forehead), jaw and blubber oils of dolphins (I-4), porpoises (5-9), the pilot whale (10), and the beluga whale (11). Apparent ly only this small group of marine mammals out of the entire animal kingdom is able to biosynthesize fatty oils containing this unusual fat ty acid. Recent biological studies by Norris (12,13) indicate that the fat ty tissues in the melon and jaw of dolphins, porpoises and toothed whales

Ipresent address: Department of Biochemistry, Nelson Biological Laboratories, Rutgers-The State University, New Brunswick, New Jersey 08903.

(i.e., the suborder Odontocet i ) may play a major role in the echolocation system of these animals. It seems appropriate, therefore, to conduct a detailed study of Odontocet i head oils to determine if their unusual chemical structure might possibly be related to their role in echolocation.

Lovern (7) and Varanasi and Malins (6) have reported that isovaleric acid occurs in mixed acid triglycerides of the melon, jaw and body oils from the porpoise Phocoena phocoena. On the other hand, Tsujimoto and Koyanagi (10) found both cetyl isovalerate and mixed isoval- eroylqong chain triglycerides in the head oil of the pilot whale Globicephala macrorhyncha. Varanasi and Matins (5) have also identified isovalerate wax esters in Tursiops gilli jaw oil. This paper reports a detailed comparison of molecular species of triglycerides found in the blubber and melon oils of the Arctic beluga whale, Delphinapterus leucas.

EXPERIMENTAL PROCEDURES

Materials

Beluga melon oil (Delphinapterus leucas) was supplied by Churchill Whale Products Ltd., Churchill, Manitoba. It came from the com- bined melons of several whales caught in Hudson Bay in August 1966. Beluga blubber oil was obtained from commercial production by the same company.

Gas Liquid Chromatography of Fatty Acids

Triglycerides were converted to methyl esters by heating in a screw-cap (Teflon fined) centri- fuge tube with 5 ml 7% (w/v) BF3/CH3OH reagent per 100 mg as described by Morrison and Smith (14). Hexane rather than benzene was added to promote sample solubilization, since benzene was found to produce an artifact with a retention time on butanediolsuccinate polyester (BDS) columns very similar to that of methyl isobutyrate. Esters were normally con- centrated into the hexane layer for gas liquid chromatography (GLC) analysis by the addit ion of 1.5 ml water to the reaction tube. Occasional direct GLC study of homogeneous reaction mixtures was a t tempted to check the quantita-

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B E L U G A W H A L E T R I G L Y C E R I D E S 675

T A B L E I

Fa t t y Acid C o m p o s i t i o n o f t he Blubber and Melon Oils F r o m the Beluga Whale

Blubber oil Melon oil

F a t t y acid Weight % Mole % Weight % Mole %

Sa t u r a t ed Iso 5 :0

9:0 Iso 10:0

10:0 Iso 11:0 An te i so 11 : 0

11:0 Iso 12:0

12:0 Iso 13:0 Ante i so 13: 0

13:0 Iso 14:0

14:0 4 , 8 , 1 2 - T r i m e t h y l t r i d e c a n o i c Iso 15:0 An te i so 15: 0 U n k n o w n

15:0 Iso 16:0 Pristanic

16:0 Iso 17:0 Ante i so 17:0

17:0 3 P h y t a n i c l

Iso 18:0 18:0 19:0 20 :0

Tota l s a tu ra t ed

M o n o u n s a t u r a t e d 12:1 0 .39 14:1607 0 .38 14:160? 0 .50 14:1607 0 .76 15:1 Trace U n k n o w n 0 .63 16:1o99 1.11 16:16o7 20 .12 16:1605 0.31 17:16o8 0 .13 18: 16ol 1+9 15.57 18:1607 4 .29 18:16o5 0 .60 19:16o9 0.01 19:1608 0.01 19:1607 0 .03 20:160 11+9 9.32 20:16o7 0 .58 20:1 605 0 .04 22:16013+11 3.42 22:1 609 1.07 22:1607 0.12 24:1609 0.21

Tota l m o n o u n s a t u r a t e d 59 .60

( C o n t i n u e d

5.80 13.15 40 .57 60 .09 . . . . . . 0 .10 0 .10

0 .03 0.05 0 .08 0 .07 0 .10 0 .13 0.25 0.22 0 .10 0 .13 1.38 1.19 0 .03 0 .05 Trace Trace 0 .05 0 .08 0 .06 0.05 0 .10 0 .13 0 .56 0.45 1.00 1.24 1.29 1.03 0.25 0 .32 2 .50 1.89 0 .03 0 .03 Trace Trace 0 .08 O. 11 O. 10 0 .07 0 .29 0.32 1.79 1.27 6.81 7 .39 6.81 4 .83 0.31 0 .34 . . . . . 0 .64 0 .66 14.64 9 .80 0 .10 0.11 0 .48 0 .33 . . . . . . 0 .21 0 .14 0 .33 0 .34 0 .54 0 .36 0 .17 0 .16 2 .06 1.31 0 .12 0 .10 . . . . . . 8.41 8.18 5.53 3.53 0 .18 0 .16 0 .62 0.38 0 .08 0 .08 0 .13 0 .09

0 .52 0 .47 0 .39 0 .24

0 .07 0.05 0.01 0.01 1.51 1.34 0 .17 0.12

Trace Trace Trace Trace 0 .04 0 .03 0 .07 0 .03

27 .15 35.15 80 .34 87 .60

0 .47 0 .24 0.19 0 .47 0 .39 0 .28 0 .55 0.39 0 .28 0 .84 0.39 0 .28

Trace 0 .49 0 .33 0 .60 0 .24 0.15 1.08 1.09 0.71

19.73 10.95 7.02 0.32 0 .09 0.05 0 .13 0 .04 0.02

13.81 2 .96 1.72 3.81 0 .62 0 .36 0 .53 0 .28 0.15 0.01 . . . . . . 0.01 . . . . . 0 .03 . . . . . . 7 .53 0 .52 0.28 0 .47 0 .03 0.02 0 .03 Trace Trace 2.55 0 .12 0.05 0 .79 0 .02 0.02 0 .08 . . . . . . 0 .16 . . . . . .

54 .00 18 .86 11.91

on fo l lowing page)

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676 C. L I T C H F I E L D , R . G . A C K M A N , J .C. S I P O S A N D C.A. E A T O N

T A B L E I ( C o n t i n u e d f r o m f o l l o w i n g page)

P o l y u n s a t u r a t e d 1 6 : 2 6 6 6 0 . 0 3 0 . 0 3 0 . 0 4 0 . 0 3 1 6 : 2 6 6 4 0 . 3 3 0 . 3 2 0 . 1 3 0 . 0 8 1 6 : 3 6 o 4 0 . 0 3 0 . 0 2 . . . . . . 1 6 : 3 6 6 3 0 . 0 9 0 . 0 8 0 . 0 2 0 . 0 2 1 6 : 4 6 6 3 0 . 1 7 0 . 1 6 0 . 0 8 0 . 0 5 16 :4661 0 . 1 2 0 . 1 3 0 . 0 7 0 . 0 5 1 8 : 2 6 o 6 1 .43 1 .29 0 . 1 9 0 . 1 0 1 8 : 3 6 6 6 0 . 0 5 0 . 0 5 0 . 0 2 0 . 0 2 1 8 : 3 6 6 3 0 . 1 4 0 . 1 3 0 . 0 2 0 . 0 2 1 8 : 4 6 o 3 0 . 2 5 0 . 2 4 . . . . . 2 0 : 2 6 6 6 0 . 1 0 0 . 0 8 0 . 0 3 0 . 0 2 2 0 : 3 6 0 6 0 .01 Trace - - - - 2 0 : 3 6 o 3 T r a c e T race - - - - 2 0 : 4 6 6 6 0 . 3 3 0 . 2 6 0 . 0 3 0 . 0 2 2 0 : 4 6 o 3 0 . 3 0 0 . 2 4 - - - - 2 0 : 5 6 o 3 3 . 8 8 3 . 2 4 0 . 1 6 0 . 0 8 21 : 5662(?) T r a c e T race - - - - 2 2 : 4 6 6 6 Trace Trace - - - - 2 2 : 5 666 T r a c e Trace - - - - 2 2 : 5 6 6 3 1.91 1 .45 - - - - 2 2 : 6 6 6 3 4 . 0 8 3 .13 - - - -

T o t a l p o l y u n s a t u r a t e d 13 .25 10 .85 0 . 7 9 0 . 4 9

1 0 0 . 0 0 1 0 0 . 0 0 9 9 . 9 9 1 0 0 . 0 0

tion of the C8-C 12 acids. The apparatus employed for GLC determina-

tion of all longer chain length fatty acids was a Perkin-Elmer Model 226 gas chromatograph fitted with a flame ionization detector (FID) and a stainless steel, open tubular (capillary) column 50 m long and 0.25 mm i.d. The coating was BDS polyester. Standard operating conditions were: column, 170 C; injection port, 270 C; helium carrier gas, 40 psig (giving a flow rate of 5 ml/nfin at 25 C). For study of lower molecular weight materials, an initial period of isothermal operation at 90 C was followed by a rapid temperature rise after emergence of the 10:0 peak and subsequent operation under standard conditions. This permitted accurate qualitative assessment of materials with chain lengths as low as C4; but quantitation of C 4 - C 6

material in relation to higher C16-C18 fatty acid methyl esters was erratic, and the impor- tant isovaleric acid component was therefore determined independently (see below). Peaks were identified by comparison with the reten- tion times of known standards and by graphical analysis of their retention times as previously described (15). Peak areas were determined using a Disc Instruments integrator and were corrected to weight per cent composition as described by Ackman and Sipos (16) for the FID. This data was subsequently integrated with an independent isovaleric acid determin- ation and converted to mole per cent.

Determination of Isovaleric Acid

Isovaleric acid was determined by GLC using

the internal standard technique. After qualita- tive verification of the absence of valeric acid (none was detected in beluga samples), about 100 mg of blubber or melon oil was weighed into a 100 ml volumetric flask. Five milliliters of 0.5 N KOH in n-butanol (BuOH) containing an appropriate amount of valeric acid standard was added. The mixture was heated on a steam bath for 15 min, with occasional swirling, and cooled. Five milliliters of 14% (w/v) BF3/BuOH reagent was added, followed by a further heating for 15 min to esterify the liberated acids. After cooling, distilled water was added to volume and the mixture shaken briskly. Part of the BuOH layer (3-5 ml) was transferred to a 100 ml volumetric flask, and a saturated NaC1 solution with approximately 0.25 ml petroleum ether was added to bring the ester rich layer which separated after shaking into the narrow neck of the flask for convenient removal by syringe for GLC analysis. GLC analyses were carried out on a glass column, 2 m x 3 mm i.d., packed with 5% SE-30 on 70/80 mesh Ana- krom ABS. The apparatus was a Barber-Colman Model 10, fitted with FID, operated at a column temperature of 120 C and a carrier gas pressure of 20 psig. Peak areas were determined with a Disc Instruments integrator. The iso- valeric acid content was then calculated from the ratio of the 5:0 and iso-5:0 peak areas, the amount of 5:0 added, and the weight of the original sample.

Since the triglyceride fractions recovered from preparative thin layer chromatography (TLC) separations were too small to be weighed

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BELUGA WHALE TRIGLYCERIDES 677

BELUGA

WHALE

XXX

XXV

XVV

TABLE II

Isovalerate Content of the Triglyceride Bands Separated From Hydrogenated Beluga Blubber

Triglycerides by Preparative TLC

Triglyceride Mole % bands isovalerate

Band XXX a Tracb Band XXV 30.2 Band XVV 61.0

ax, long chain fatty acid; V, isovaleric acid.

ELUGA BLUI~ER TRIGLYCERIDES

FRACTION XXX

S B B' S M M' S FRACTION XXV

FIG. 1. Separation of beluga blubber and melon triglycerides according to isovaleric acid content using TLC on silicic acid. XXX, triglycerides containing no isovaleric acid; XXV, diacyl-monoisovalerin; XVV, monoacyl-diisolvalerin; S, standard mixture of XXX,

FRACTION XVV XXV, XVV and VVV (not marked) produced by inter- �89 esterification of trimyristin and triisovalerin. B, un- - --~. hydrogenated beluga blubber oil. B', hydrogenated beluga blubber oil. M, unhydrogenated beluga melon oil. M', hydrogenated beluga melon oil. Operating conditions: 200 x 200 mm TLC plate coated with 0.25 mm layer of Adsorbosil 1; developed in 87:12:1 petroleum ether-diethylether-acetic acid; spots visual- ized by charring with H2SO4/K2Cr207 solution.

accurately, they were analyzed using bo th valeric and heptadecanoic acid internal stan- dards permit t ing comple te quan t i t a t ion in a s tep-programmed GLC analysis. F r o m 1-4 mg of sample were placed in a 50 ml vo lumet r ic flask and 1.0 ml 0.5 N K O H / B u O H containing the appropriate amounts of acid standards was added. The s toppered flask was heated on a steam bath for 10 min, and then 1.0 ml 14% (w/v) BF3 /BuOH reagent was added. The re- s toppered flask was heated for ano ther 10 min, cooled, and filled to 50 ml wi th distilled water. Then 0.25 ml pe t ro leum ether was added and the flask shaken vigorously for 1 min. Af te r separation, a sample for GLC analysis was taken directly f rom the top layer. GLC analysis o f the C5 components was carded out as before , fol lowing which the co lumn tempera tu re was rapidly raised to 200 C for the rest of the analysis. The total fa t ty acid compos i t ion of the sample was then calculated f rom the ratio of the 5:0 and iso-5:0 peak areas, the ratio of the 17:0 and C10-C22 peak areas, and the amounts of 5:0 and 17:0 originally added.

xxx xxv

x x v

FIG. 2. Separation of unhydrogenated beluga blubber oil triglycerides by permeation chromatog- raphy. Initial bands from total sample (top curve) were individually rechromatographed (three bottom curves) for further purification. Shaded areas represent fractions collected. XXX, triglycerides containing no isovaleric acid; XXV, diacyl-monoisovalerin; XVV, monoacyl-diisovalerin. Operating conditions: 25 x 420 mm column of 200/400 mesh styrene divinyl-benzene copolymer beads; * 100 mg triglycerides dissolved in 1.0 ml benzene applied to column; samples eluted with benzene at 1.3 ml/nfin; column temperature 28 C.

Al though a very small amount ('x,0.6 mole %) n'C17 acid was present in the original b lubber triglycerides, this me thod was qui te accurate enough to ident i fy the fractions o f tr iglycerides containing no isovaleric acid (XXX), diacyl- monoisovaler in (XXV), and monoacyl-di isol- valerin (XVV) separated by TLC.

Hydrogenation

To improve resolut ion in TLC and GLC, bo th melon and b lubber otis were ful ly hydrog- enated prior to tdglycer ide analysis. This was accomplished by the me thod of Farquhar et al. (17) using freshly distilled dioxane as a solvent in place of ethanol .

VLC

Hydrogena ted beluga blubber and melon lipid

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678 C. LITCHFIELD, R.G. ACKMAN, J.C. SIPOS AND C.A. EATON

BELUGA BLUBBER TRIGLYCERIOE$

48 ~

37 5~ 41 60

J BAND XXX

J ~ BAND XXV 43

BAND XVV

TABLE III

FIG. 3. Gas chromatograms of hydrogenated total beluga blubber triglycerides and of the three bands separated by TLC on silicic acid. XXX, triglycerides containing no isovaleric acid; XXV, diacyl-monoiso- valerin; XVV, monoacyl-diisovalerin. Operating con- ditions: 560 x 2.5 mm i.d. stainless steel column packed with 3.0% JXR on 100/120 mesh Gas Chrom Q; column temperature programmed 160-+350 C at 4 deg/min; 100 ml/min. He carrier gas; flash heater 350 C.

were separated by preparative TLC on 1.0 mm thick layers of silicic acid (Adsorbosi l 1, Ap- plied Science Laborator ies , State College, Pa.) impregna ted wi th 0.02% R h o d a m i n e 6G. Plates were deve loped in 87:12:1 pe t ro l eum ether- d ie thyle ther -ace t ic acid. Bands were then lo- cated under UV light, scraped in to individual ch roma tog raphy co lumns , and e luted wi th di- e thylet l ier .

Permeation Chromatography The tr iglycerides f rom 1.0 g of unhydrogen-

a ted beluga b lubber oil were e luted f rom a 20 x 100 m m co lumn of alumina (Woelm, basic activity grade 1) wi th 150 ml d ie thyle ther . Af te r solvent evapora t ion , the tr iglycerides were redissolved in 10 ml benzene and applied, 1 ml o f solut ion at a t ime, to a 25 x 420 mm s tyrene-d iv inylbenzene copo lymer bead column (Dew X2, 200 /400 mesh, lot 07153). The sample was e luted wi th addi t ional benzene at 28 C wi th a f low rate of 1.3 ml /min (18). A different ia l recording r e f r ac tome te r (19) was used to m o n i t o r the eluate. The respect ive tr iglyceride f rac t ions f r o m the 10 individual runs were combined and ch roma tog raphed once more on the same co lumn for fu r the r purifi- cat ion.

GLC of Triglycerides GLC analyses of hydrogena ted tr iglycerides

were carried out under the o p t i m u m opera t ing

Triglyceride Composition of Hydrogenated Blubber and

Melon Oils From the Beluga Whale

5, ~ Carbon Blubber oil, Melon oil, ~o~ ~ ~ ~ no. mole % mole %

�9 a 6o

"6 ~ Diisovaleroyl triglycerides (XVV) 20 --- 0.17 22 -- 1.27 23 -- 3.89 24 1.24 18.07 25 1.30 31.78 26 3.78 28.66 27 -- 0.60 28 0.64 2.27

Total XVV 6.96 86.71

Monoisovaleroyl triglycerides (XXV)

29 -- 1 31 0.29 33 0.86 34 0.28 10.49 35 2.68 36 0.53 37 5.64 38 0.31 -- 39 5.86 0.95 40 0.17 -- 41 4.17 0.30 43 2.33 0.19 45 0.86 0.13 47 0.20 0.11

Total XXV 24.18 12.17

Triglycerides with no isovaleric acid (XXX)

42 0.51 Trace 44 1.41 0.08 46 3.37 0.17 48 6.43 0.27 50 9.06 0.21 52 10.69 0.16 54 11.03 0.11 56 10.31 0.07 58 7.93 0.04 60 4.96 -- 62 2.23 -- 64 0.76 -- 66 0.17 --

Total XXX 68.86 1.11

100.00 99.99

condi t ions for marine oils descr ibed by Harlow, Li tchfield and Reiser (20). An F & M 400 gas ch roma tog raph equ ipped wi th F1D and auto- matic t empera tu re p rogramming was used. The 560 x 2.5 mm i.d. stainless steel co lumn packed wi th 3.0% JXR on 100/120 mesh Gas Chrom Q was p rog rammed f rom 160 C to 350 C at 4 deg/min wi th 100 ml /min He carrier gas.

Peaks were ident i f ied as to carbon n u m b e r by coch ro ma t o g rap h y wi th k n o w n compounds . Trimyris t in , t r ipalmit in , t r is tearin, t r iarachidin

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BELUGA WHALE TRIGLYCERIDES 679

and tribehenenin of 99% purity (Applied Sci- ence Laboratories, State College, Pa.) were used to identify the carbon numbers of triglycerides containing no isovaleric acid. Mixtures of tri- stearin and triisovaterin (Eastman Organic Chemicals, Rochester, N.Y.), tripalmitin and triisovalerin, and trimyristin and triisovalerin were interesterified with a NaOCH 3 catalyst (21) to produce standards of isovaleroyl- triglycerides. Interesterification of trimyristin and triisovalerin, for example, produced a mixture of trimyristin, isovalero-dimyristin, di- isovalero-myristin and triisovalerin that was used to identify the peaks of 15, 24 ,33 and 42 carbon number. Peak areas were determined by planimetry. Quantitative response factors for the various carbon number triglycerides were determined from known composition mixtures of trihexanoin, trioctanoin, tridecanoin, trilau- rin, trimyristin, tripalmitin, tristearin, triarach- idin and tribehenin. The fm values (molar calibration factors determined by the internal normalization technique) (22) of the simple triglycerides in the calibration mixtures were then plotted vs. carbon number, and fm values for all carbon numbers were interpolated from the resulting graph. Triglyceride compositions are reported in mole per cent.

RESULTS AND DISCUSSION

Fatty Acid Composition

Since beluga blubber and melon oils are known to contain isovaleric acid, the first step was to determine the general fatty acid compo- sition of our samples. GLC analysis of the fatty acids gave the results reported in Table I. The melon oil contains a very high level (60.1 mole %) of isovaleric acid, substantial amounts of long chain branched acids (16.9%), and very little highly polyunsaturated material (0.5%). The blubber oil contains less isovaleric (13.2%), fewer long chain branched acids (2.7%), and appreciable amounts (10.9%) of the polyun- saturated acids typical of marine oils.

The types and amounts of long chain branched acids in the blubber oil are similar to those found in most marine depot lipids (23,24); only in the melon oil do they accumu- late to an unusual degree. Most of the long chain branched acids are of the iso series, indicating their possible genesis from isovaleric acid. The levels of isovaleric acid reported here for beluga melon and blubber oils are almost twice those found by Williams and Maslov (11). Assuming their analyses to be reasonably accu- rate, it would appear that the isovaleric acid content of the beluga whale is subject to considerable variation. Although of circumpolar

6E'I_UGA MELON TRIGLYCER[OES

~s

FIG. 4. Gas chromatograms of hydrogenated total beluga melon triglycerides. Column was overloaded in lower chromatogram so that minor peaks could be quantitated. Operating conditions same as for Figure 3.

distribution, the genus Delphinapterus has but a single species, so this difference in isovaleric content cannot be attributed to species differ- ences.

TLC

Beluga melon and blubber oils were next examined for lipid class composition by TLC on silicic acid. Both samples could be separated into three main spots in an 87:12:1 petroleum ether-diethylether-acetic acid solvent system (Fig. 1). Cochromatography with trimyristin, isovalero-dimyristin, diisovalero-myristin and triisovalerin showed that these three spots correspond to triglyceride molecules containing 0, 1 and 2 isovaleroyl moieties. The greatest distance up the plate was covered by XXX; XXV moved somewhat less; while XVV moved the shortest distance. No triisovalerin could be detected by TLC. Resolution of the three spots was significantly improved by hydrogenation of the samples prior to TLC.

Further evidence for the identify of the isovaleroyl-triglycerides was obtained by recov- ering the XXX, XXV, and XVV bands from a preparative TLC separation of hydrogenated beluga blubber oil and determining the fatty acid composition of each fraction. The results (Table II) show the expected compositions. Fraction XXX contains essentially no isovaleric acid; fraction XXV contains about one third isovaleric acid on a molar basis; and fraction XVV is approximately two thirds isovaleric. Slight deviations from theoretical isovalerate content are probably due to incomplete separa- tion of the three bands.

The resolution of triglycerides containing 0, 1, 2 and 3 isovaleric acid moieties is not completely unexpected. Other workers such as Blank and Privett (25) and Kleiman et al. (26) have shown that triglycerides containing one acetic or butyric acid moiety can be easily separated from long chain triglycerides by TLC on silicic acid. Certainly this type of separation

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680 c. LITCHFIELD, R.G. ACKMAN, J.C. SIPOS AND C.A. EATON

is extremely useful with cetacean triglycerides containing isovaleric acid, since it allows rapid, easy fractionation of these complex mixtures according to isovaleric acid content.

Permeation Chromatography

Resolution of beluga blubber triglycerides by permeation chromatography on a polystyrene gel column yielded three fractions (Fig. 2). After rechromatography for further enrich- ment, GLC of the intact triglycerides from each fraction clearly identified them as the XXX, XXV and the XVV bands already found by TLC. This resolution of XXX, XXV and XVV triglycerides by permeation chromatography would generally be expected, since this tech- nique separates molecules mainly on the basis of molecular weight (27). The resolution of the XXX, XXV and XVV fractions by permeation chromatography is approximately the same as by TLC on silicic acid, provided sample size and enhancement of resolution by hydrogenation are considered.

GLC

Natural triglycerides mixtures containing short chain fatty acids can be fractionated into a large number of peaks by GLC (28,29). Figure 3 (top) shows the resolution of the hydrogenated blubber oil triglycerides by this technique into a phenomenal 30 peaks. These peaks seem to be arranged into three groupings: C24-C28 , C31-C45 and C42-C66. The identities of these three groups of beluga blubber triglyc- erides were clarified by GLC analysis of the XXX, XXV and XVV bands that had been isolated from hydrogenated beluga blubber triglycerides by preparative TLC. Band XXX contains only C42-C66 triglycerides of even carbon number (Fig. 3) corresponding to tri- glyceride molecules containing no isovaleric acid. Band XXV contains mostly odd carbon number C31-C45 peaks, which are triglycerides having one isovaleroyl moiety per molecule. Band XVV contains C24-C28 material, which must be monoacyl-diisovalerins. Thus, these chromatograms provide additional evidence for the identity of the XXX, XXV and XVV bands tentatively identified by their TLC Rf values and their fatty acid compositions. The large differences in triglyceride retention times at- tributable to the presence of 0, 1 or 2 isovaleric acid chains allow complete GLC quantitation of the XXX, XXV and XVV fractions without prior subfractionation by TLC on silicic acid. XXV molecules are mainly odd carbon number triglycerides and thus have different elution temperatures from the XXX species, which are mostly even carbon number triglycerides. Quan-

ti tation of the chromatograms (Table III) shows that beluga blubber oil contains 68.9 mole % XXX, 24.2% XXV, and 7.0% XVV. No triiso- valerin could be detected by GLC, even when the starting column temperature was lowered to 100 C.

GLC analysis of hydrogenated beluga melon triglycerides (Fig. 4) shows them to be com- posed of mostly XVV species from C23 through C 28. Injection of an extra large sample indicates that XXV and XXX species are indeed present but only in minor amounts. No peak was observed at the temperature where syn- thetic triisovalerin eluted from the column. Monoacyl-diisovalerin constitutes 86.7 mote % of the melon triglycerides (Table III); very little XXV (12.2%) and XXX (1.1%) are present. The high level of branched-chain acids in beluga melon triglycerides leads to shouldering on many of the peaks. Note that the C24 and C26 peaks both exhibit front shoulders, probably due to the presence of both iso- and n-long chain acids in the XVV molecules. These shoulders could also be due to the presence of triglyceride positional isomers (29), but the presence of high levels of iso- 14: 0 and iso- 16:0 acids makes the former explanation consider- ably more probable. The C29-C37 area of the beluga melon triglycerides is extremely com- plex on the chromatogram in Figure 4. The presence of both short and long chain branched acids in the same XXV molecules probably accounts for this complexity. Thus accurate carbon number assignments could not be made for the C29-C37 peaks, and they were quanti- tared as a single group for reporting in Table III.

Comparison of average fatty acid chain length values (20) indicates that the carbon number distributions reported in Table III are quite close to the correct values. The triglyceride data for the blubber oil gives an average acyl chain length of 15.99 vs. 15.63 calculated from the fatty acid composition. The respective values for the melon oil are 8.85 and 9.01.

Implications for Cetacean Echolocation

Norris (12,13) and Wood (30) have postu- lated that the melon of fatty tissue in the forehead of the beluga whale and other Odon- toceti (dolphins, porpoises and toothed whales) functions as a sound transducer and possibly a sonic lens in the echolocation process of these animals. The sound transmitter for echoloca- tion apparently lies in the nasal passages, immediately behind the melon. This sound is picked up by the fatty tissue in the melon and directionally transmitted to the seawater in front of the head. A fatty oil is closely

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BELUGA WHALE TRIGLYCERIDES 681

i m p e d a n c e - m a t c h e d to seawater so t h a t the energy losses dur ing the t r ans fe r o f s o u n d waves f r o m m e l o n to seawate r are very low (13) . The presen t work ind ica tes t h a t a t r ig lycer ide , monoacy l -d i i sova le r in , cons t i t u t e s 86.7 mole % of the me lon oil in the be luga whale . The long chain acyl m o i e t y of th is t r ig lycer ide is p r imar- ily iso a n d s t ra ight cha in C11-C17 f a t t y acids. Many an imals o t h e r t h a n r u m i n a n t s p r o b a b l y p roduce m i n o r a m o u n t s of isovaleric acid. H u m a n b lood , for example , con ta ins a b o u t 194 /~g/100 ml p lasma (31) . The O d o n t o c e t i are the excep t iona l an imals w h i c h exp lo i t th is acid by i n c o r p o r a t i n g i t i n to the i r h e a d and b l u b b e r oils. The presence of the acid in the b l u b b e r oils is poss ibly due to spi l lover of t he t r ig lycer ides syn thes ized for the e c h o l o c a t i o n sys tem in t he head .

Gi lmore (32) has suggested t h a t isovaleric acid is n e e d e d t o m a i n t a i n t he m e l o n f a t t y oi l in a l iquid s ta te at the e x t r e m e l y low envi ron- m e n t a l t e m p e r a t u r e s ( d o w n t o -2 C) in w h i c h these an imals live. The low t e m p e r a t u r e requi re- m e n t is possibly due t o a pauc i ty of b l o o d vessels in the skin over ly ing t he me lon , as those migh t in t e r fe re w i t h h igh f r e q u e n c y s o u n d t r ansmiss ion i n to the water , and a t e m p e r a t u r e gradient may exis t across the me lon . Ord ina ry mar ine oils, inc lud ing those of m a n y fish and of the Myst ice t i , are sub jec t to f o r m a t i o n of sohds (s tear ine) at t e m p e r a t u r e s in some cases as h igh as 20 C (discussed t echn ica l ly as cold clearing; cf. AOCS c loud po in t tes t ) (33) . In our exper i ence beluga melon oil is clear d o w n to well be low 0 C.

It seems p r e m a t u r e to advance any h y p o t h e - sis re la t ing l ipid c o m p o s i t i o n to e c h o l o c a t i o n abi l i ty in the O d o n t o c e t i un t i l we have con- s iderably more i n f o r m a t i o n on the precise me lon and j aw lipids f o u n d in these an imals as well as wha t unusua l acous t ica l p roper t i e s , if any, these i sovaler ic -conta in ing oils migh t pos- sess. F u r t h e r e x p e r i m e n t s a long these l ines are cu r ren t ly in progress.

ACKNOWLEDGMENTS

This investigation was supported in part by grants from the National Institutes of Health (AM-06011) and from the Industrial Development Service, Depart- ment of Fisheries and Forestry, Government of Canada. The cooperation of Churchill Whale Products, Ltd. and the advice of scientists of the Fisheries Research Board of Canada Arctic Unit have been most valuable.

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