Bulletin of the Japanese Society of Scientific Fisheries Vol. 35, No. 7, 1969 653
The Hydrocarbons of Shark Liver Oils
Mitsu KAYAMA*,Yasuhiko TSUCHIYA**,and Judd C. NEVENZEL*** (ReceivedMarch 31, 1969)
Almost a half century ago, TSUJIMOTOand others reported the existence of highly un saturated1-3), monounsaturated4) and saturated hydrocarbons5,6) in some shark liver oils and named those hydrocarbons squalene1) (or spinacene2)), zamene4) and pristanee6) res pectively. Later the chemical structures were assiduously investigated by many workers, and finally squalene was determined to be 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22- tetracosahexaene7-10), "zamene", 2, 6, 10, 14-tetramethyl-1-pentadecene11) and its isomeric monoolefins12),and pristane (norphytane), 2, 6, 10, 14-tetramethylpentadecane13). Squalene is now recognized as an important intermediate in the biosynthesis of choles terol since Langdon and Bloch proved its role14). The questions still remain from the present study, however, why certain deep-sea sharks contain a great quantity of squalene and little cholesterol in the liver oil, and why squalene is accompanied by a considerable amount of pristane and a trace amount of "zamene" and phytadienesls15). It is of interest, therefore, to investigate the hydrocarbon content and composition of the various shark liver oils by the recently developed techniques, and to determine the chromatographic behavior and infrared spectra of squalene, squalane (hydrogenated squalene), pristane, "zamene" and phytadienes as a basis for further biochemical studies.
Experimental and Results
Shark liver oils. The shark livers of Triakis scyllia*, Squalus acanthias**, Apristurus macrorhinchus***, Centroscyllium ritteri****, Centrophorus spp.***** and Cetorhinus maximus****** were used as sources of the liver oils. Triakis was caught in Matsushima Bay, Squalus, Apristurus and Centroscyllium were trawled off the coast of Sanriku, Cen trophorus was caught by long line in Suruga Bay, and Cetorhinus was harpooned off southern California.
* Dept . of Fisheries, Hiroshima University, Fukuyama, Japan(広 島 大 学 水 畜 産 学 部 水 産 学 科) ** Dept , of Fisheries, Tohoku University, Sendai, Japan(東 北 大 学 農 学 部 水 産 学 科) *** Laboratory of Nuclear Medicine and Radiation Biology , University of California, Los Angeles, U. S. A. * Leopard shark , dochi-zame in Japanese. ** Dogfish , abura (tsuno)-zame in Japanese. *** Nagahera -zame in Japanese . **** Kasumi -zame in Japanese . ***** Shiratsubu -zame in Japanese, one of the three kinds of ai-zame. ****** Basking shark , uba-zame in Japanese. 654
The whole or partial livers were ground in a blendor with five to ten times their volumes
of chloroform-methanol (2:1, V/V). The filtered extracts were washed with water, dried
over anhydrous MgSO4, and the solvent was removed in a rotary evaporator to give the
total liver oils. The oil content and some of the oil characteristics are included in Table 2.
Saponification of liver oil. One volume of oil was hydrolyzed in ten volumes of 5%
alcoholic KOH under reflux on a steam bath for 1.5 hours. The hydrolysate was diluted
with an equal volume of water and first extracted with two portions of petroleum ether
(b. p. 35 to 60•Ž); the remainder was diluted again with an equal volume of water and finally
extracted twice with diethyl ether. The petroleum ether and diethyl ether extracts were
pooled and washed with 50% alcohol and water successively, dehydrated with anhydrous
MgSO4, and dried under a nitrogen stream.
Silicic acid column chromatography. The liver oil and its unsaponifiable materials
were fractionated quantitatively into various lipid classes by adsorption column chroma
tography on silicic acid16).
The silicic acid (Mallinckrodt, 100 mesh) was mixed with Celite 545 (Johns-Manville)
in the proportions of 9:1 (W/W) and packed dry into a column measuring 2.0cm•~9.5cm
(1 column volume•à20ml). After activation by prewashing with 2 column volumes
each of acetone, diethyl ether and petroleum ether sequentially, the sample (100 to 200mg)
dissolved in a small quantity of petroleum ether was loaded on the column, and the various
lipid types eluted with 5 column volumes each of petroleum ether, 2%, 5%, 17.5% and
22.5% diethyl ether in petroleum ether (V/V), diethyl ether and methanol, using a slight
vacuum to increase the flow rate. Representative chromatograms of the shark liver oils
and of their unsaponifiable materials are shown in Fig. 1.
The main components were obtained as follows: the hydrocarbons were eluted with
petroleum ether, the esters of cholesterol, vitamin A and fatty alcohols with 2% diethyl ether,
most of the diacyl glyceryl ethers and triglycerides with 5% diethyl ether, the free acids and
alcohols with 17.5% diethyl ether, the sterols with 22.5% diethyl ether, the glyceryl ethers and
the monoglycerides with diethy ether, and the phospholipids with methanol17,18). Although
the highest amount appeared in the triglyceride and diacyl glyceryl ether fractions (5% diethyl
ether fraction) of the Triakis, Squalus and Apristurus liver oils , surprisingly large quantities
of the hydrocarbons (petroleum ether fraction) are contained in the Centroscyllium , Centro phorus and Cetorhinus liver oils. On the other hand, for the unsaponifiable materials the
largest fraction is found in the sterol fraction of Triakis , in the glyceryl ether fractions of
Squalus and Apristurus, and in the hydrocarbon fractions of Centroscyllium , Centrophorus and Cetorhinus. It is apparent from these data that the contents not only of hydrocarbons
but also of other components in the shark liver oils vary greatly , depending on the species used. Thin-layer chromatography. Thin-layer chromatography (TLC) on silica gel plates
was used for the resolution and assay of the lipid classes of the shark liver oil17) with special 655
Fig. 1. Silicic acid chromatograms of shark liver oils (left) and their unsaponifiable materials (right). Each bar represents the total weight of material eluted from a 2cm•~9.5cm column by five column volumes (100ml) each of the solvents indicated on the horizontal axis. Abbreviations: P. E., petroleum ether; 2, 5, 17.5 and 22.5 are the percentages of diethyl ether in petroleum ether (V/V); Et2O, diethyl ether; MeOH, methanol. reference to the hydrocarbons. Although the hydrocarbon class consisting of squalene , pristane, "zamene" and phytadienes was not easily resolved on the silicic acid column, fairly good separations were obtained on thin layers of silica gel impregnated with silver nitrate19) 656
by using appropriate solvents. Glass plates (20cm•~20cm) were coated with a layer
about 250-300ƒÊ in thickness of silica gel (Merck reagent 7729, with 5% of plaster of Paris
added as binder) or Silica Gel G (Merck reagent 7731), impregnated or not impregnated
with 5% of silver nitrate in the usual way, and dried at 120•Ž for 30 minutes.
Fig. 2. Thin-layer chromatograms of shark liver oil hydrocarbons on silica gel
plates. Plates A and B were not and plate C was impregnated with 5% AgNO3 . Solvents: plate A, petroleum ether-diethyl ether-acetic acid, 90:10:1 (V/ V/V); plates B and C, n-hexane. Detection: 50% sulfuric acid spray followed by charring or 2•L, 7•L-dichlorofluorescein spray followed by ultraviolet detection . S amples: 1, Centroscyllium liver oil; 2 & 7, petroleum ether fraction of Apris turus unsaponifiable materials; 3 & 8 , pristane fraction obtained by vacuum distillation of 2 & 7; 4 & 9, pristane (isolated by preparative GLC and hydro
genated); 5 & 10, squalane; 6 & 11, squalene; 12 & 13 , "zamene" and phy tadienes, respectively, separated on TLC impregnated with AgNO 3 as shown at 8.
The results of TLC are presented in Fig . 2. The first plate (A) illustrates the chroma togram of the Centroscyllium liver oil as the representative one on a silica gel plate developed
with the solvent system , petroleum ether-diethyl ether-acetic acid (90:10:1 , V/V/V)20). From the solvent front to the origin there were fractionated the hydroca rbons, esters, diacyl glyceryl ethers17,21), triglycerides, free fatty acids and probably traces of alcohols , cholesterol, partial glycerides, and phospholipids. Subfractionation within the hydrocarbon class of
interest was not achieved by this procedure . The second plate (B) shows the chromatograms of the Apristurus hydrocarbon fraction , which was separated by the column chromatographic
procedure, and the reference hydrocarbons on a silica gel plate developed with n -hexane. The resolution between pristane and squalene was satisfactory , but pristane, "zamene" and squalane were almost coincident . By charring at 110•Ž after spraying with 50% aq . sulfuric acid, squalene was visible first , developing a maroon color; the fully saturated pristane and squalane showed only a slight darkening after still longer heating at this tem perature. "Zamene", which was associated with pristane in trace amounts , was located on the lower part of the pristane spot , staining more deeply than pristane. In an attempt to improve the separation of the saturated , monounsaturated, diunsaturated and polyunsa 657
turated hydrocarbons, we tirst tried the mercuric acetate addition method22). However, attempts to prepare the acetoxymercuri-methoxy compounds of the unsaturated hydrocar bons resulted in failure, because such hydrocarbons did not dissolve in methanol. We next tried argentation TLC19). For the preparation and impregnation of plates, 23.75g of silica gel containing 5% of the binder were mixed thoroughly with 55ml of water in which
1.25g of silver nitrate was dissolved. Fig. 2 (C) shows a plate of silica gel impregnated with 5% silver nitrate and developed with n-hexane. Using this procedure we succeeded finally in separating pristane and "zamene". Either by sulfuric acid charring or by 2•L,7•L- dichiorofluoreseein (0.2% in alcohol) spraying followed by ultraviolet detection, pristane appeared just below the solvent front, squalene near the origin and "zamene" a little below the midpoint. It is noteworthy that a greater separation was obtained between pristane and "zamene" by argentation TLC, even in the case of a great quantity of pristane and a trace amount of "zamene", than by gas-liquid chromatography (in which pristane tailing
overlapped a small peak of "zamene") and that one more spot, phytadienes, was detected between "zamene" and squalene as shown in the third plate (C). The hydrogenated "za
mene", which was separated by TLC impregnated with silver nitrate, behaved as pristane in TLC as well as in gas-liquid chromatography.
Gas-liquid chromatography. Gas-liquid chromatography (GLC) was carried out to get the composition of the hydrocarbons and to determine the GLC behavior of those compounds. The instrument used was a Shimadzu 2B 101 (Shimadzu Instrument Co.,
Kyoto) equipped with a hydrogen flame ionization detector. Three stationary phases,
ethyleneglycol succinate polyester (EGS, synthesized without catalyt), Apiezon M (Ap. M,
Associated Electrical Industries Ltd.) and silicone rubber gum SE-30* (SE-30, General
Electric Co.) coated on 80/100 mesh Diasolid L (Nihon Chromato Kogyo, Tokyo) and
80/100 mesh Chromosorb W (Johns-Manville) were used; the specific columns and operat
ing conditions used are described in Table 1. The liquid phase loadings are given as initial
weight percentages on the supporting materials.
As standard samples, pristane and squalene were isolated from the hydrocarbon frac
tions of Apristurus and Centrophorus liver oils using preparative GLC; squalane was obtained
by hydrogenation of squalene; and "zamene" and phytadienes were separated by prepara
tive TLC on AgNO3-impregnated silica gel from a vacuum distilled Apristurus pristane
fraction which contained 1% of "zamene" and lesser amounts of phytadienes. A suitable
mixture of these hydrocarbons was prepared, and its gas-liquid chromatographic behavior
on several different columns was determined. The results are given in Table 1. All the
retention times in Table 1 were measured to the leading edge of each peak, which gave values
which were nearly constant and independent of the sample size. The retention times mea
* Presented by Prof . J. F. Mead, Laboratory of Nuclear Medicine and Radiation Biology, University of California, Los Angeles, California. 658
Table 1. Retention times of hydrocarbons obtained from shark liver oil.
All the retention times were measured at the starting points of elution curves * C . olumn and condition: 1) 15% EGS 3m , 198•Ž, 54ml/min; 2) 15% EGS 3m, 189•Ž, 48ml/min; 3) 15% EGS 3m, 151•Ž, 52.5ml/min; 4) 1.5% Ap. M 1m, 198•Ž , 60ml/min; 5) 1.5% Ap. M 1m, 189•Ž , 60ml/min; 6) 1.5% Ap. M 1m, 151•Ž, 60ml/min; 7) 1 .5% SE-30lm, 198•Ž, 60ml/min; 8) 1.5% SE-30 1m, 189•Ž , 60ml/min; 9) 1.5% SE-30 1m, 151•Ž, 60ml/min .** P ristane and "zamene" peaks not resolved . *** Pristane and "zamene" peak s incompletely resolved .* *** These values were derived from th e data of 6) and 9) on semi-logarithmic linear relations at 151•Ž .Abb reviations: EGS, ethyleneglycol succinate (coated on Diasolid L) ; Ap. M, Apiezon M (coatedCh on romosorb W); SE-30, silicone rubber gum SE -30 (coated on Chromosorb W) . sured at the peak maxima , however, varied considerably with the sample size , owing to di stortion and skewing of the peaks , especially with the larger samples of squalane and squalene, which have long retention times . Pristane, "zamene", and phytadienes were eluted very rapidly under the GLC conditions used , and separations among the three com pounds were better on the EGS column than on the non-polar columns . The order of 659 appearanceof squalane and squalene was not reversed on the polar and non-polar columns used. The carbon numbers23)of these hydrocarbons relative to n-fatty acid methyl esters (NationalInstitutes of Health Standard Mixture E and F)* and n-hydrocarbons (C14,C16 and C18,over 99% purity)** were calculated to aid in the structural information. The contents of hydrocarbons and sterols. In Table 2 are listed the contents of un saponifiablematerials, hydrocarbons, and sterols in the liver oils. The hydrocarbon values werederived from the petroleum ether fractions eluted in silicic acid chromatography, as shownin Fig. 1. The pristane and squalene values were obtained by GLC analysis; how ever,as was mentioned above, the pristane peak in GLC actually included some "zamene", amountingto 1% of the pristane fraction vacuum distilled from Apristurus liver oil. More meaningfulcomparisons of the amounts of pristane and sterols among the various species can be made by eliminating the effect of the widely differing amounts of squalene present. Thereforesome values in Table 2 are expressed as "g per 100g fresh liver tissue, lipid-free". This is admittedly an artificial basis, but an accessible way of correcting for the very large amounts of lipid present in some of these livers. Table 2. Sharkliver oils and theirhydrocarbon and sterolcontents.
* Distributed by the Lipid Program of National Institutes of Health, Bethesda, Maryland. ** Presented by Prof . S. Seto, Institute of Non-Aqueous Solution, Tohoku University. 660
The percentages of hydrocarbons in the oils from the different species vary 160-fold, but squalene is the principal hydrocarbon in every case. The Triakis, Squalus and Apris turus oils contain small percentages of total hydrocarbons, of which pristane is a major constituent (over 6%). On the other hand, in the liver oils of Centroscyllium, Centrophorus and Cetorhinus the hydrocarbons are 34-64% of the total oil, and Squalene is almost the sole component. However, if we express the pristane content as a percentage of the fresh liver tissue, it is present in highest amounts in Centroscyllium, Apristurus and Cetorhinus (5.0%, 2.1% and 1.6%, respectively). In Squalus pristane is much less important, at less than 0.3%. The amounts of squalene present in Centroscyllium, Centrophorus and Cetor hinus are truly remarkable, being 5-15% of the total body weight. The ratios of hydrocar bons to sterols range from 0.1 to 88; however, this variation is seen to be almost entirely due to the widely different squalene contents of the different species, since expressed as the percent of the fresh liver tissue (lipid-free) the sterol values only range from 2.1 to 7.4 (a factor of 3.5), while the squalene contents vary by a factor of over 1,500. Infrared spectra. The infrared spectra were measured in Nippon Bunko infrared spectrometer (Japan Spectroscopic Co., Ltd., Tokyo), Model IR-S. The substances were examined in the liquid state between NaCl plates at room temperature . For the comparison of the hydrocarbon fractions of the liver oils used, the infrared spectra of all the fractions were measured. The most striking differences were found at 1670cm-1 due to stretching vibrations of the non-conjugated double bonds, and at 1365- 1385cm-1 24,25)owing to the isopropyl group doublet . The spectra of Centroscyllium, Centrophorus and Cetorhinus are very similar to the curve of squalene. With increasing content of pristane (as in the case of the Triakis, Squalus and Apristurus hydrocarbon frac tions) the spectra resemble the sum of the squalene and pristane curves. In Fig. 3 are shown the infrared spectra of (A) pristane , (B) "zamene", (C) mixed phytadienes, and (D) squalene, all isolated from shark liver oils as described above, and of (E) squalane, prepared by hydrogenation of authentic squalene. Pristane can be distin guished by the splitting of the 1380cm-1 band of deformation frequencies into two com ponents, one at 1383cm-1 and the other at 1368cm-1 ("isopropyl splitting"), and by the band at 1170cm-1 with a shoulder at about 1150cm-1, due to skeletal vibrations of the iso propyl groups27). On the other hand, the spectrum of squalene is characterized by the bands due to the carbon-carbon double bonds, such as the stretching vibrations of the non conjugated double bonds at 1670cm-1 and the out-of-plane deformations at 980 and 830 cm-1 of the terminal (CH3)2C=and the CH3R1C=CHR2 groupings28). The spectrum of squalane is very similar to that of pristane . "Zamene" is clearly distinguished from the saturated hydrocarbon, pristane, by the C=C stretching mode of an unconjugated olefinat 1645cm-1, the characteristic out-of-plane deformation at 885cm-1 of the vinylidene group, R1R2C=CH224),and a decrease relative to pristane of the-isopropyl splitting at 1380cm-1. 661
Fig. 3. Infrared spectra of shark liver hydrocarbons. Spectrum A, pristane; 13, "zamene"; C, phytadienes; D, squalene; E, squalane.
Phytadienes,which have the structures of unsymmetrical conjugated dienes15),are charater izedby the stretching vibrations at 1640 and 1610cm-1 25).
Discussion It would be interest to relate these results to the ecology of the various species. The sharks used may be grouped according to the depth at which they commonly live. Triakis migrates into shallow water bays, and Squalus also lives near the surface. Apristurus dwells at comparatively moderate depths and Centroscyllium in rather deeper waters. Centrophoruslives in deep water and is caught at 600 to 800m or often more. Cetorhinus, the so-calledbasking shark, is caught at the surface; however, there is some evidence that it may winter in deep water. Physical factors such as pressure, temperature, salinity, and dissolvedoxygen vary with the depth. Thirty years ago TSUJIMOTO29)classified shark liver oilsinto two groups, with the dividing line at a specific gravity of d1540.9000, and noted that the content of hydrocarbons (mainly squalene) varied inversely as the specific gravity; his ideaswere reaffirmed by HIGASHI,KANEKO, and SUGII30).The data of Table 2 are in ex cellentagreement with TSUJIMOTO'shypothesis and add weight to the theory that in general the deep-sea sharks contain much larger amounts of hydrocarbons than do shallow water species. We do not know the reason for the high content of hydrocarbons, but it seems probablethat their functions are for buoyancy in such fish lacking a swimbladder, morever, mayby for gas solubility28), and for the pressure and cold resistances in deep sea. Note 662
that the squalene contents of Centroscyllium, Centrophorus and Cetorhinus correspond to 5-15% of the total body weight. Some of the deep-sea bony fishes have been found to contain comparable percentages of wax esters in their muscle tissues31,32). Squalene is an intermediate in the biosynthesis of cholesterol, and so it would be of great significance to know how deep-sea sharks accumulate very large amounts of squalene while maintaining only slightly elevated cholesterol levels. Investigating the biosynthesis from mevalonic acid of squalene and cholesterol by rat liver enzyme preparations , POPJAK et al.33)observed that under anaerobic conditions only squalene was synthesized, and a reducing agent was not required; the squalene formed anaerobically could be cyclizedto sterol after admission of air to the incubation flasks by repeated additions of NAD and NADP. It is conceivable, therefore, that in the deep-sea sharks cholesterogenesis may be blocked just beyond the step of squalene synthesis because of the lack of oxygen or other factors, including the coenzymes required for cyclization. Recently it was pointed out that 2, 3-oxidosqualene is the key intermediate for cyclization to sterols34). In our preli minary experiment it was found that aerobic condition was essential for the normal acetate to-cholesterol pathway in carp and rainbow trout homogenates . The contents of sterols, in every case over 90% cholesterol, were not significantly different among the shark species used, when they were expressed on a lipid-free fresh liver weight basis, even though there are extraordinary differences in the squalene contents . The values found for total sterols, 2.1 to 7.4g per 100g liver tissue, are comparable to other shark liver data35,36)and about twice as high as those of land animals37). From this point of view, in the deep-sea shark's liver the enzyme systems leading to squalene seem to be increased disproportionately compared to the capacity for squalene cyclization, and the latter becomes the rate-determining step. Control of squalene biosyn thesis by a feed-back signal from the increasing level of squalene present seems to be im perfect (probably because of the low solubility of this non-polar hydrocarbon in the aqueous media), and it steadily accumulates, soon forming a second phase of oil droplets. At this stage the bulk of the squalene in the liver becomes metabolically inert. The hydrocarbons pristane, "zamene", and phytadienes are closely related structurally to the diterpene phytol, as suggested by SORENSENand SORENSEN13). According to BLUMER et al.38)pristane is derived largely, if not exclusively, from Calanus sp. S of copepods and once formed is very stable, passing up the food chain very nearly unchanged. AVIGANand BLUMER39)demonstrated that labelled phytol (in nature derived from the chlorophyll of the phytoplankton at the base of the food chain) when fed to Calanus gave rise to labelled pristane. If BLUMER'Stheory is correct, for non-plankton feeders such as Squalus (and possibly Apristurus and Centroscyllium, although less is known about their feeding habits), the fish or other organisms consumed must contain at least the same total amount of pristane found in the shark. In this case there would be one or more intermediate organisms in 663
thefood chain between the Calanus copepods and Squalus, each of which passes along the pristanewith no degradation and with minimal losses. It seems probable that additional organismswill be found which degrade phytol via pristane and thus introduce this hydrocar bon into the food chain of sharks.
Summary The hydrocarbon fractions of the liver oils of six sharks, Triakis scyllia, Squalus acan thias,Apristurus macrorhinchus, Centroscyllium ritteri, Centrophorus spp. and Cetorhinus maximus,were separated by silicic acid chromatography. The hydrocarbon compositions wereanalyzed by gas-liquid chromatography. The content and composition of hydrocar bons vary with the species used. The liver oils of Triakis, Squalus and Apristurus contain small amounts of hydrocarbons in which a comparatively high amount of pristane is con tained as well as squalene. On the contrary, the liver oils of Centroscyllium, Centrophorus and Cetorhinus contain a great quantity of hydrocarbons consisting almost entirely of squalene with a trace amount of pristane. By using silver nitrate impregnated adsorbents in TLC (argentation TLC) "zamene" and phytadienes, which are associated with pristane in trace amounts, were separated from the distilled pristane fraction. The chromatographic behavior of the isolated pristane, "zamene" , phytadienes, squalene, and squalane and their infrared spectra were determined. The biochemical cyclization of squalene to sterols in the shark livers rich in lipids of high squalene content and the biogenesis of pristane are discussed. Moreover, it is sug gestedthat the primary function of the extraordinarily high content of hydrocarbons in someshark livers may be for buoyancy in such deep-sea fishes lacking swimbladders.
Acknowledgements This work was partly supported by a grant from the Ministry of Education of Japan. One of the authors (JCN) was supported by contract AT (04-1) GEN-12 between the U.S. Atomic Energy Commission and the University of California. The grateful acknowledgements are made to the following people for the gifts of the sharks and shark livers: Mr. Y. TAKAHASHI,Head of the Matsushima Aquarium, for Triakis scyllia samples; Mr. S. MUTO,Tohoku Regional Fisheries Research Laboratory, for Squalus acanthias livers; Mr. K. MATSUMOTO,Yaizu Marine Chemical Co., for Cen trophorusspp. liver; Mr. K. HAYASHI,Graduate Student of Tohoku University (present: Tohoku Regional Fisheries Research Laboratory), for Apristurus macrorhinchus and Cen troscylliumritteri samples; Dr. S. APPLEGATEand Miss S. HOWELLS,Los Angeles County Museum of Natural History, for Cetorhinus maximus liver oil. Thanks are also due to Dr. H. SONEand Mr. T. ARAWAKA,Riken Vitamin Oil Co., Ltd., for the gift of the squalene and pristane concentrates. The authors wish to express their gratitude to Prof. James F. 664
MEAD, Laboratory of Nuclear Medicine and Radiation Biology, University of California, Los Angeles, for his kind criticism.
References
1) M. TSUJIMOTO: J. Ind. Eng. Chem. Japan, 9, 953 (1906); 19, 277-280 (1916); 20, 953-1017, 1069-1098 (1917); 21, 1015-1042 (1918); J. Ind. Eng. Chem., 8, 889-896 (1916). 2) A. C. CHAPMAN: J. Chem. Soc., Trans., 111, 56-69 (1917); 113, 458-466 (1918). 3) I. M. HEILBRON,E. D. KAMM,and W. M. OWENS: J. Chem. Soc., 1630-1644 (1926). 4) M. TSUJIMOTO: J. Chem. Soc. Japan, 55, 702-704 (1934); Bull. Chem. Soc. Japan, 10, 149-153 (1935). 5) M. TSUJIMOTO: J. Ind. Eng. Chem. Japan, 20, 1099-1101 (1917); J. Ind. Eng. Chem., 9, 1098-1099 (1917). 6) T. TOYAMA: J. Ind. Eng. Chem. Japan, 27, 333-350 (1924); Chem. Umschau, 30, 181-187 (1923). 7) I. M. HEILBRON,W. M. OWENS,and 1. A. SIMPSON: J. Chem. Soc., 873-883 (1929). 8) I. M. HEILERONand A. THOMPSON: ibid., 883-892 (1929). 9) P. KARRER,A. HELFENSTEIN,H. WEHRLI,and A. WETTSTEIN: Hely. Chitn. Acta,13,1084-1099 (1930). 10) P. KARRERand A. HELFENSTEIN: ibid., 14, 78-85 (1931). 11) P. K. CHRISTENSENand N. A. SoRENSEN: Acta Chem. Scand., 5, 751-756 (1951). 12) M. BLVI ER and D. W. THOMAS: Science, 148, 370-371 (1965). 13) J. S. SORENSENand N. A. SORENSEN: Acta Chem. Scand., 3, 939-944 (1949). 14) R. G. LANGOONand K. BLOCH: J. Biol. Chem., 200, 129-134, 135-144 (1953). 15) M. BLUMERand D. W. THOMAS: Science, 147, 1148-1149 (1965). 16) D. L. FILLERUPand J. F. MEAD: Proc. Soc. Exptl. Biol. Med., 83, 574-577 (1953). 17) M. KAYAMAand Y. TSUCHIYA: Tohoku J. Agr. Res., 15, 259-267 (1964). 18) M. KAYAMAand Y. TSUCHIYA: This Bull., 34, 640-645, 646-650 (1968). 19) L. J. MORRIS: in "New Biochemical Separations" (A. T. JAMESand L. J. MORRIS,ed.), 259-319, D. Van Nostrand Co., Ltd., London (1964). 20) H. K. MANGOLDand D. C. MALINS: J. AIn. Oil Chem. Soc., 37, 383-385 (1960). 21) D. C. MALINS,J. C. WEKELL,and C . R. HOULE: J. Lipid Res., 6, 100 (1965). 22) E. JANTZENand H. ANDREAS: Chem. Ber., 92, 1427-1437 (1959). 23) F. P. WOODFORDand C. M. VAN GENT: J. Lipid Res., 1, 188-190 (1960). 24) L. J. BELLAMY: The Infra-red Spectra of Complex Molecules , Methuen and Co., Ltd., London (1958). Also John Wiley & Sons, New York (1958) . 25) R. M. SILVERSTEINand G. C. BASSLER: Spectrometric Identification of Organic Compounds , 2nd ed., 78-81, John Wiley & Sons , New York (1967). 26) Y. ISIUK.AwA: J. Fac. Fish. &. Animal. Husb., Hiroshima Univ., 3, 351-374 (1961). 27) J. PLIVAand N. A. SORENSEN: Acta Chem. Scand., 4, 846-849 (1950). 28) H. W. THOMPSONand P. TORKINGTON: Tr . Faraday Soc., 41, 246-260 (1945). 29) M. TSUMMMOro:Study of Liver Oil, 189-190 , Maruzen Co., Ltd., Tokyo (1936). 30) H. HIGASHI,T. KANEKO,and K . SuGn: This Bull., 20,633-640(1954). 31) J. C. NEVENZEL,W. RODEGKER , and J. F. MEAD: Biochemistry, 4, 1589-1594 (1965).32)M . MORI, T. SAITO,and Y. NAKANISHI: This Bull ., 32, 668-672 (1966). 33) G. POPJAK,L. GOSSELIN, .I Y. GORE, and R . G. GOULD: Biochem. J., 69, 238-248 (1958). 34) E. J. COREY,W. E . RUSSEY,and P. R. ORTIZ DE MONTELLANO: J . Ain. Chem. Soc., 80, 4750-4751 (1966). 35) M. L. KARNOVSKY,W. S. RAPSON, H. M. SCHWARTZ,M. BLACK,and N. J. VANRENSBURG: J. SOC. Chem. Ind., 67, 104-107 (1948). 36) Y. SHIMMAand H. SHIMMA: Bull. Tokai Reg. Fish. Res. Lab., No. 48, 53-61 (1966).37) R. P. COOK: in "Cholesterol; Chemistry , Biochemistry, and Pathology" (R. P. COOK, ed.), 149, 152 , 159, Academic Press, New York (1958). 38) M. BLUMER,M, M . MULLIN,and D. W. THOMAS: Science , 140, 974 (1963).39)J . AVIGANand M. BLUMER: J. Lipid Res., 9, 350-352 (1968) .