675

Fatty Acids and Fatty Alcohols of Wax Esters in the Orange Roughy: Specific Textures of Minor Polyunsaturated and Branched-Chain Components TORU TAKAGI* , YUTAKA ITABASHI and SHINGO ASO, Department of Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate, Japan, 041

ABSTRACT

Open-tubular gas chromatography was carried out on fatty acids and alcohols obtained from wax esters of the orange roughy, Hoplostethus atlanticus, caught at sea off New Zealand. The major (above 5%) components were 16:1(n-7), 18: l(n-9) and (n-7), 20:1(n-9) and (n-7), and 22:1(n-11, n-13) as fatty acids, and 16:0, 18:0, 18: l(n-9), 20: l(n-9) and (n-7), and 22:1 (n-11, n-13) as fatty alcohols. The total percentages of the minor components were 10% in the acids and 26% in the alcohols. The 22:1/ 20:1 ratio of the fatty alcohols obtained in this study was less than 1.0, although the ratio for the Atlantic orange roughy has been reported as being greater than 1.0. The contents of polyenes were as low as 2.48% in the acids and 0.95% in the alcohols, but their compositions showed some specific fea- tures. The percentages of the C~6-C22 dienes in the total polyenes were remarkably high, 57.7% of these acids and 53.1% of these alcohols. The most important dienes were 18:2(n-6) in the acids and 20: 2(n-6) in the alcohols. Lipids 20:675-679, 1985.

INTRODUCTION

The fatty oil of the deep-sea teleost, the orange roughy, contains wax esters as the major components. It could be used as a substitute for sperm whale off and jojoba oil (1). Mori et al. reported the fatty acid and alcohol compositions Of wax esters from the flesh of Hoplostethus gilchristi caught at 1000 m depth off New Zea- land in 1978 (2). Subsequently, Hayashi and Takagi reported the acid and alcohol composi- tions of wax esters from the flesh of H. atlanti- cus caught in the same waters (3). Since then, four papers (4-7) have been published on the compositions of orange roughy wax esters. In these studies, the major components, saturated and monounsaturated straight-chain acids and alcohols, were the main subjects of investiga- tion.

The present paper also describes the detailed compositions of the minor polyunsaturated and branched-chain fatty acids and alcohols from wax esters of orange roughy caught off New Zealand. In addition, the compositions of the positional isomers of the monounsaturated com- ponents are reported for comparison with those shown in previous papers (6,7).

MATERIALS AND METHODS

Materials and Separation of Wax Esters The sample oil was produced, with meal,

from orange roughy caught in deep water off New Zealand by a trawler in 1983. The crude

*To whom correspondence should be addressed.

oil obtained from Nikko Chemicals Co., Tokyo, in February, 1984 was stored at about -20 C for 3 mo until used in this study.

Separation of wax esters from the oil was accomplished by thin layer chromatography (TLC) with Silica Gel G coated at 0.5 mm in thickness, using benzene/n-hexane (3:2, v/v) as a developing solvent. Rhodamine 6G solution in ethanol was used as a visualizing reagent.

Preparation of Methyl Esters and Acetates

The wax esters were converted to fatty acid methyl esters and fatty alcohols by direct trans- esterification with 5% HCI in methanol/benzene (1:2, v/v) heated at 80 C in screw-cap test tubes for 3 hr under nitrogen. The methyl esters and alcohols were separated by TLC with Silica Gel G plates with development in n-hexane/ ether (85 : 15, v/v). The alcohol fraction was con- verted into acetates by heating with acetic an- hydride/pyridine (1:1, v/v) at 80 C in screw-cap centrifuge tubes for 1 hr under nitrogen.

Gas Liquid Chromatography (GLC)

Fatty acid and alcohol compositions were obtained by GLC of the methyl esters and ace- tates using a Shimadzu GC 6 AMPF instrument (Shimadzu Seisakusho Co., K y o t o ) e q u i p p e d with a dual FID detector and a glass capillary WCOT column (50 m x 0.28 m m i d ) coated with SP 2300 (Supelco Inc., Bellefonte, Penn- sylvania). The carrier gas was H2 with a flow rate of 0.5 ml/min. The column temperature was 200 C, with the detector and sample inlet

LIPIDS, VOL. 20, NO. 10 (1985)

676 T. T A K A G I , Y . I T A B A S H I A N D S. A S O

T A B L E 1

C o m p o s i t i o n o f F a t t y A c i d s a n d A l c o h o l s o f O r a n g e R o u g h y W a x E s t e r s

T h i s s t u d y P r e v i o u s s t u d y ( 3 ) a

C o m p o n e n t A c i d A l c o h o l C o m p o n e n t A c i d A l c o h o l

1 3 : 0 0 . 0 4 0 . 0 3 1 3 : 0 -- - 1 4 : 0 0 . 9 3 2 . 0 8 1 4 : 0 1 .4 2.1 1 5 : 0 0 . 0 5 0 , 5 4 1 5 : 0 0 .1 0 .7 1 6 : 0 0 . 8 1 2 2 . 1 6 1 6 : 0 2 . 1 2 8 . 3 1 7 : 0 0 . 5 0 0 , 4 9 1 7 : 0 0 . 4 0 .2 1 8 : 0 0 . 2 8 5 . 9 5 1 8 : 0 0 . 7 7 .6 1 9 : 0 -- 0 , 1 7 1 9 : 0 -- 0 .2 2 0 : 0 0 . 0 3 0 . 5 2 2 0 : 0 -- 0 .5 2 1 : 0 -- 0 . 0 6 2 1 : 0 -- -- 2 2 : 0 -- 0 , 1 2 2 2 : 0 -- 0 .2 1 5 : 0 iso -- 0 . 1 6 1 5 : 0 i so -- 0 .2

a n t e i s o 0 . 0 3 0 , 0 3 a n t e i s o 0.1 -- 1 6 : 0 i so 0 . 0 1 0 . 2 4 1 6 : 0 i so -- 0 . 4

a n t e i s o -- 0 . 0 2 a n t e i s o -- -- 1 7 : 0 i s o -- 0 . 5 8 1 7 : 0 i so 0 . 3 0 . 3

a n t e i s o -- 0 . 3 2 a n t e i s o - 0 . 8 1 8 : 0 i so -- 1 , 0 5 1 8 : 0 i so -- 0 .5 1 9 : 0 iso -- 0 . 0 4 1 9 : 0 i so -- -- 2 0 : 0 i so -- 0 . 2 3 2 0 : 0 i so -- --

a n t e i s o -- -- a n t e i s o 0 . 2 0 .2

T o t a l ( s a t . ) 2 . 6 8 3 4 . 7 8 5 .5 4 2 . 0

1 4 : 1 (n -5 ) 0 . 3 3 -- 1 6 : 1 (n -9 ) -- 0 , 0 9

(n -7 ) 1 1 . 5 4 0 , 7 8 1 7 : 1 (n -8 ) 0 . 7 3 0 . 3 0 1 8 : 1 (n -9 ) 5 1 . 1 1 1 1 , 3 5

( n - 7 ) 4 . 9 1 2 . 7 6 (n -5 ) 0 . 1 2 0 . 4 7

1 9 : 1 (n -8 ) 0 . 2 9 0 . 3 3 2 0 : 1 (n -9 ) 1 1 . 5 6 1 6 . 1 7

( n - 7 ) 5 . 5 4 9 . 1 0 (n -5 ) 0 . 6 8 1 . 2 3

2 2 : 1 ( n - I 1) 5 . 8 7 1 2 , 9 8 (n -9 ) 1 . 4 3 3 . 8 0

2 3 : 1 - b _ 0 . 1 1 2 4 : 1 ( n - I 1) -- 0 . 5 1

(n -9 ) 0 . 6 7 2 . 9 7

T o t a l ( m o n o e n e ) 9 4 . 8 4 6 4 . 2 6

1 4 : 1 0 . 3 -- 1 6 : 1 13 .4 1.9

1 7 : 1 0 .5 0 .5 1 8 : 1 5 9 . 4 16 .2

1 9 : 1 0 . 6 0 .5 2 0 : 1 12 .4 17.1

2 2 : 1 5 .4 14 .5

m

_ m

9 2 . 6 5 3 . 9

1 6 : 2 (n -6 ) 0 . 0 8 -- 1 7 : 2 -- 0.1 1 8 : 2 (n -6 ) 1 . 0 0 0 . 1 2 1 8 : 2 1 .3 0 .8

(n -3 ) 0 . 0 5 . . . . 1 8 : 3 (n -6 ) 0 . 0 4 -- 1 8 : 3 0 . 4 --

( n - 3 ) 0 . 2 0 0 . 0 9 1 9 : 2 -- 0 .2 1 8 : 4 (n -3 ) 0 . 1 8 . . . . 2 0 : 2 (n -6 ) 0 . 1 4 0 . 2 8 2 0 : 2 0 .1 1 .8 2 0 : 3 (n -6 ) 0 . 0 5 . . . .

( n - a ) 0 . 1 3 . . . . 2 0 : 4 (n -6 ) 0 . 0 8 0 . 1 8 2 0 : 4 0.1 --

( n - a ) 0 . 1 9 0 . 0 3 -- - - 2 0 : 5 (n -3 ) 0 . 2 1 . . . . 2 2 : 2 (11-6) 0 . 0 4 0 . 1 1 2 2 : 2 -- 1.1 2 2 : 5 (n -3 ) 0 . 0 3 0 . 0 2 -- -- -- 2 2 : 6 ( n - 3 ) 0 . 0 6 0 . 1 3 2 4 : 2 -- 0.1

T o t a l ( p o l y e n e ) 2 . 4 8 0 . 9 6 1 .9 4.1

a W a x e s t e r s w e r e s e p a r a t e d f r o m t h e t o t a l l i p i d s e x t r a c t e d f r o m t h e m u s c l e o f a m a l e H. a t lan t icus c a u g h t f r o m ca. 9 4 0 m d e p t h o f f N e w Z e a l a n d . T h e e x t r a c t i o n w a s d o n e b y m e t h o d o f Bl igh a n d D y e r . G L C w a s d o n e on 1.5 m X 3 m m c o l u m n s p a c k e d w i t h 1 0 % D E G S o n C h r o m o s o r b W a n d w i t h 5% S I L A R 10 C o n G a s C h r o m Q.

b T e n t a t i v e l y i d e n t i f i e d b y p l o t t i n g o f c a r b o n n u m b e r s vs. l o g r e l a t i v e r e t e n t i o n t i m e s f o r t h e n -8 s e r i e s o f m o n o e n o i c a c i d m e t h y l e s t e r s .

L I P I D S , V O L . 20, N O ; 10 (1985)

ORANGE ROUGHY WAX ESTERS 677

at 230 C. Peak area pe rcen tages were o b t a i n e d wi th a Sh imadzu in t eg ra to r C-R2AX.

The c o m p o n e n t of each peak was iden t i f i ed as s h o w n in Table I o n t he basis of ag reemen t of t he r e t e n t i o n data w i th those of re fe rence , m e t h y l esters of sea u rch in f a t ty acids (8,9) , and ace ta tes of f a t ty a lcohols o b t a i n e d by LiA1H 4 r e d u c t i o n of the same m e t h y l esters. The log plot p rocedure and t he sys temat ic sepa- r a t ion fac to r p rocedure (10) were used concur - r en t ly for the iden t i f i ca t ion .

Argentation-TLC (AgNOa-TLC)

The f a t t y acid m e t h y l esters and a lcohol ace- ta tes also were f r ac t i ona t ed accord ing to the i r degree of u n s a t u r a t i o n o n silver n i t ra te - impreg- na t ed layers of Silica Gel G b y developing w i th e thy l a c e t a t e / n - h e x a n e (1 :9 , v/v).

Hydrogenation of Methyl Esters and Acetates

The m e t h y l esters and aceta tes were com- p le te ly h y d r o g e n a t e d b y the usual p rocedures descr ibed in a previous paper (3), using 5% palla- d ium on ca rbon as a catalyst .

AgNO 3-TLC Analysis with the Chromarod- latroscan System

The p rocedures descr ibed in previous papers (11-13) were used for the analysis. Chromarods - SII c leaned by be ing passed t h r o u g h a f lame were immersed in a 2.5% so lu t ion of silver n i t r a t e in ace ton i t r i l e for 15 min , and t h e n acti- vated b y hea t ing in an oven at 120 C for 3 hr. The rods were spo t t ed wi th 1 pl of 1-10% sample so lu t ion in c h l o r o f o r m , and deve loped wi th e t h y l a c e t a t e / h e x a n e (0 .5 :9 .5 , v/v) for the sepa ra t ion of sa turates , m o n o e n e s , dienes and o the r polyenes . Af te r developing, the rods were air-dried and t h e n scanned wi th a f l ame ioniza- t ion de t ec to r in an I a t roscan TH-10 i n s t r u m e n t ( I a t roscan Labora tor ies , Tokyo) . Peak areas were measu red wi th a C h r o m a t o p a c C-R1A (Shi- m a d z u Seisakusho).

Saturated Acids and Alcohols

A low p r o p o r t i o n o f s a tu ra t ed acids is a char- acter is t ic of the wax esters f rom orange r o u g h y oils. The c o n t e n t of sa tu ra ted acids in the t o t a l acids has n o t exceeded 5% in any of the previ- ous papers (1-7), and the figure was less t h a n 1% in th is s tudy . The c o n t e n t of sa tu ra ted alco- hols o b t a i n e d in this s tudy was marked ly lower t h a n t ha t r e p o r t e d in our previous paper (3), as s h o w n in Table 1, while the m o n o u n s a t u r a t e d a lcohol c o n t e n t in this s tudy was r e m a r k a b l y higher. The acid and a lcohol compos i t i ons in this s t udy were s o m e w h a t ana logous to those r epo r t ed b y Mori e t al. (2). The 1 6 : 0 / 1 8 : 0 ra t ios r epo r t ed for t he a lcohols of o range r o u g h y wax esters in t he l i t e ra tu re are in the range o f 3 to 5, excep t for a f igure of less t h a n 1.0 in t he data r e p o r t e d by Buisson et al. (1). The ra t io was a b o u t 4 in this s tudy .

The c o n t e n t of b r a n c h e d c o m p o n e n t s was greater in the a lcohols t h a n in the acids. The t o t a l c o n t e n t of b r a n c h e d sa tu ra ted a lcohols was 2.6% of the t o t a l a lcohols . We verif ied this b y t o t a l h y d r o g e n a t i o n and reanalysis . The de- ta i led c o m p o s i t i o n of h y d r o g e n a t e d ace ta tes of the a lcohols is s h o w n in Table 2. The compar i - son of the u n h y d r o g e n a t e d c o m p o n e n t ( f r o m Table 1) and of the t o t a l co r r e spond ing c o m p o - nen t s a f te r h y d r o g e n a t i o n , wi th in wh ich the re is a 2.4% c o n t e n t of b r a n c h e d a lcohols , con-

TABLE 2

Composition of H y d r o g e n a t e d P r o d u c t s o f F a t t y A c i d s

a n d F a t t y Alcohols

F o u n d

Component Acid Alcohol

13:0 14:0 15:0 16:0 17:0

R ESU LTS AND DISCUSSION 18:0 19:0

Major Fatty Acid and Alcohol Components 20:0

F a t t y acids and a lcohols f o u n d as c o m p o - 21:0 22:0 nen t s of wax esters in the orange r o u g h y oil by 23:o WCOT GLC are s h o w n in Table 1. There were 24:0 six ma jo r (above 5%) c o m p o n e n t s in each case. iso-15:0 They were 16:1(n-7) , 18 :1 (n-9) and (n-7) , 20:1 ai:15:0

iso- 1 6 : 0 (n-9) and (n-7), 22 :1 (n -11 , n-13) as f a t t y acids, ai-16:o and 16:0, 18:0, 18 :1(n-9) , 2 0 : l ( n - 9 ) a n d (n-7), iso-17:o and 22 :1 (n -11 , n-13) as f a t t y alcohols. The ai-17:0 to ta l c o n t e n t s of the ma jo r c o m p o n e n t s , 90% iso-18:o

i s o - 1 9 : 0 for the acids and 74% for the a lcohols , show iso-2o:o t ha t the p r o p o r t i o n of m i n o r c o m p o n e n t s was h igher in the alcohols.

C a l c u l a t e d a

A c i d Alcohol

0.07 0.50 0.04 0.03 0.97 1.83 1.26 2.08

- 0.53 0.05 0.54 11.67 22.00 12.34 23.03

0.85 0.86 1.23 0.79 56.30 20.54 57.80 20.61

0.68 0.92 0.29 0.50 19.25 27.87 18.85 27.65

0.30 0.43 -- 0.09 8.02 18.57 7.44 18.48

- - - - - - 0.11 1.17 3.57 0.67 3.43

- 0 . 1 7 - - 0.16 -- 0.13 0.03 0.03 - - 0.25 0.01 0.24 - - -- - 0.02

0.72 0.48 -- 0.58 - 0.28 -- 0.32 - 0.78 -- 1.05 -- 0.14 - 0.04 - 0.15 -- 0.23

a C a l c u l a t e d from the data in Table 1.

LIPIDS, VOL. 20, NO. 10 0985)

678 T. TAKAGI, Y. ITABASHI AND S. ASO

f i r m s that the branched alcohols in the wax esters are mostly saturated. Except for one re- port (3), an appreciable content of branched alcohols has not been reported in any previous paper on orange roughy oils (1,2,4-7). However, capillary GLC has shown more branched chain alcohols than acids in wax esters of the white barracudina Paralepsis rissoi Kr~yeri Bonaparte 1840 from Nova Scotian waters (14).

Monounsaturated Acids and Alcohols

The monoenes, 16:1(n-7), 18:1(n-9) and (n-7), 20:l(n-11), 22:1(n-11), were reported as being the major components of the acids and alcohols in the analysis of orange roughy wax esters by Sargent et al. (7). Additional minor monoenes, 18:1(n-5), 20:1(n-7) and (n-5), 22:1 (n-9) and (n-7), and 24:1(n-9), were detected by WCOT GLC in this study. Occurrence of 24:1(n-11) and (n-13) was reported by Body et al. (6) from the results of gas liquid chroma- tography-mass spectroscopy of di(trimethylsilyl) ethers of diols which were obtained by oxida- tion, but they were not detected in this study. Under the conditions of the WCOT GLC used in this study, the 22:1 (n-13) component was in- cluded with the 22:1(n-11) recorded in Table 1.

The 22:1/20:1 ratios in the fatty alcohols were found to be greater than 1.0 in Atlantic orange roughy wax esters by Sargent et al. (7). In contrast, the 22:1/20:1 ratios are 1.0 or less in Pacific orange roughy wax esters (1-6). The ratio obtained in this study also was obviously less than 1.0. This variation of 22:1/20:1 alco- hol ratios might depend on the biosynthetic mechanisms of chain elongation and desatura- tion of shorter acids and alcohols, de novo syn- thesis of 20:1 and 22:1 acids and their conver- sion to alcohols, and the catabolism of the acids and alcohols as energy sources, as described in the literature (7,15). The compositions of monounsaturated fatty alcohols in certain fish body lipids and commercial fish oils were com- pared with those in copepods, and 22:1 alcohols in the wax esters of copepods are assumed to be precursors of the 22:1 fatty acids of fish depot fats, specifically of the dominant 22:1(n-11) isomer (16). In this study, the major 22:1 acid in the wax esters was 22:1(n-11) acid, and it may be formed mainly from 22:1(n-11) alcohol. Therefore, the high proportions of 22:1/20:1 of the alcohols compared to those of the acids reported in the literature (1-6), and obtained in this study, are assumed to be due to the low conversion of the 22:1 alcohols to the 22:1 acids in the orange roughy.

High n-6/n-3 Ratios of Polyunsaturated Acids and Alcohols

Compositions of polyunsaturated acids and alcohols in orange roughy wax esters have been reported in only a few papers (2,3,7), probably due to their low content. In this study, the de- tailed composition of the polyunsaturated com- ponents was obtained by WCOT GLC as shown in Table 1. The results showed some specific features which were different from the polyun- saturated components of ordinary marine fish oils (triacylglycerols) where n-3 fatty acids are dominant. The proportions of n-6 components apparently were higher in the polyunsaturated acids and alcohols of the orange roughy used in this study. The alcohols favor 18:2(n-6), 20:2 (n-6), 22:2(n-6), and 20:4(n-6) relative to the C20 and C22 n-3 series, except for the relatively important 22:6(n-3). On the contrary, the acids favor the Cls and C20 n-3 series relative to the CIs and C2o n-6 series, except for 18:2(n-6), as shown in Table 1.

Abundance of Dienes in Polyunsaturated Acids and Alcohols

The second specific feature of the polyun- saturated acids and alcohols of orange roughy wax esters is their high proportion of diunsatu- rated components, as shown in Table 1. Dienoic acids, such as 16:2(n-6), 18:2(n-6) and (n-3), and 20:2(n-6) and (n-3), have been found as minor components of marine fish oils, but they do not seem to accumulate in fish to the extent of more than 1 or 2% of the total fatty acids (17,18), and the dienoic acid content is below 10% in the polyenoic acids. The percentages of the dienes in the polyunsaturated acids and alcohols of the orange roughy wax esters were at usually high levels, 57.7 and 53.1%, respec- tively, in this study. Linoleic acid was the most important of the dienoic acids, while 20:2(n-6) alcohol was the most important diunsaturated alcohol. Linoleic acid [18:2(n-6)] was the only dienoic acid found in wax esters of the orange roughy caught off the west coast of Britain (7), and its percentage in the total of polyenoic acids was 33%. The diene fraction of the fatty acid methyl esters and alcohol acetates sepa- rated by AgNOa-TLC revealed peaks listed as the dienes in Table 1. Percentages of the unsatu, rates in acids and alcohols obtained by AgNOa- TLC by Chromarod-Iatroscan are shown in Table 3. This independent method supports the abundance of diunsaturated components in the polyunsaturated acids and alcohols of orange roughy wax esters.

LIPIDS, VOL. 20, NO. 10 (1985)

ORANGE ROUGHY WAX ESTERS 679

TABLE 3

AgNO 3 -TLC Analysis of Polyenoic Acids and Alcohols Based on the Unsaturation with an Iatroscan

Fatty acid % Fatty alcohol %

Found Calcd. a Found Calcd. a

Dienes 50.9 52.8 46.8 53.1 Trienes _b _ 8.1 9.4 Other polyenes 49.1 c 47.2 45.1 37.5

aCalculated from the data in Table 1. bThe trienoate peak for acids could not be sepa-

rated from the more highly unsaturated polyenoate peak.

Clncluding percentages of the trienoates.

ACKNOWLEDGMENT

R.G. Ackman, Technical University of Nova Scotia, and Takashi Kaneda, Tohoku University, provided their advice.

REFERENCES

1. Buisson, D.H., Body, D.R., Dougherty, G.J., Eyres, L., and Vlieg, P. (1982) J. Am. Oil Chem. Soc. 59,390-395.

2. Mori, M., Yasuda, S., and Nishimuro, S. (1978) Bull. Jpn. Soc. Fish. 44,363-367.

3. Hayashi, K., and Takagi, T. (1980) Bull. Jpn. Soc. Sci. Fish. 46, 459-463.

4. Grigor, M.R., Thomas, C.R., Jones, P.D., and Buisson, D.H. (1983) Lipids 18, 585-588.

5. Buisson, D.H. (1983) The Future of Fish Oils in New Zealand, in Proc. Intern. Conf. on Oils, Fats and Waxes, Auckland, pp. 29-34, Duromark Pub., Auckland, New Zealand.

6. Body, D.R., Johnson,C.B., and Shaw, G.J. ( i983) Monounsaturated Substituents of Marine Wax Esters, in Proc. Intern. Conf. on Oils, Fats and Waxes, Auckland, pp. 188-189, 192, Duromark Pub., Auckland, New Zealand.

7. Sargent, J.R., Gatten, R.R., and Merrett, N.R. (1983) Mar. Biol. 74,281-286.

8. Itabashi, Y., and Takagi, T. (1980) Yukagaku 29, 855-865.

9. Ackman, R.G. (1983) Some Old and New Prob- lems in Capillary Chromatography, in Proc. In- tern. Conf. on Oils, Fats and Waxes, Auckland, pp. 176-179, Duromark Pub., Auckland, New Zealand.

10. Ackman, R.G. (1969) in Methods in Enzymology (Lowenstein, I.M., ed.) Vol. 14, pp. 329-381, Academic Press, New York, NY.

11. Tanaka, M., Itoh, T., and Kaneko, H. (1979) Yukagaku 28, 22-25.

12. Sebedio, J.L., and Ackman, R.G. (1981) J. Chro- matogr. Sci. 19,552-557.

13. Sebedio, J.L., Farquharson, T.E., and Ackman, R.G. (1982) Lipids 17,469-475.

14. Ackman, R.G., Hooper, S.N., Epstein, S., and Kelleher, M. (1972) J. Am. Oil Chem. Soc. 49, 378-382.

15. Sargent, J.R., Lee, R.F., and Nevenzel, J.C. (1976) in Marine Waxes, in Chemistry and Bio- chemistry of Natural Waxes (Kolattukudy, P.E., ed.) pp. 59-91, Elsevier, Amsterdam.

16. Ratnayake, W.N., and Ackman, R.G. (1979) Lipids 14,795-803.

17. Ackman, R.G. (1980) Fish Lipids. Part 1, in Ad- vances in Fish Sciences and Technology (Connell, J.J., ed.) pp. 86-103, Fishing News Books, Garn- ham, Surrey, England.

18. Ackman, R.G. (1982) Fatty Acid Compositions of Fish Oils, in Nutritional Evaluation of Long Chain Fatty Acids in Fish Oil (Barlow, S.M., and Stansby, M.E., eds.) pp. 25-88, Academic Press, New York, NY.

[ Received F e b r u a r y 19, 1985 ]

L1PIDS, VOL. 20, NO. 10 (1985)