The lipid biochemistry of marine organisms

THE LIPID BIOCHEMISTRY OF

M A R I N E O R G A N I S M S

DONALD C, MALINS and JOHN C. WEKELL

U.S. Fish and Wildlife Service, Department of the Interior, Seattle, Washington

I. INTRODUCTION

Marine animals, unlike their terrestrial counterparts, are intimately exposed to fluctuating environmental conditions, such as oxygen concentration, temperature, and pressure. Furthermore, to add complexity, other forces related to the peculiar ecology of the marine world, such as the food chain and symbiotic relationships, are also intimately associated with the biochemistry of marine species. Such variables involve the modification and regulation, to a greater or lesser extent, of fundamental biochemical processes. Accordingly, the tasks before us will be to discuss the biochemistry of lipids in the light of those factors that are peculiar to the marine environment. Because Lovern 5° and Malins ~ have reviewed the structure and composition of marine lipids, it is our hope that the present approach will offer an interesting and unique perspective.

II. COMPOSITION, STRUCI'URE, AND METABOLISM

A. Hydrocarbons A variety of hydrocarbons are found in marine organisms. Some species

contain only trace amounts, whereas the lipids of some sharks comprise almost entirely hydrocarbons. 2s, 41 For example, the hepatic lipids of the elasmo- branch fish Centrophorus uyato contain 90 % squalene. 2s In the marine biosphere this hydrocarbon appears to predominate, but small amounts of pristane (2,6,10,14-tetramethylpentadecane) and its unsaturated analogue norphytene (2,6,10,14-tetramethylpentadec,-l-ene) are usually present. I, 5, 15 Lambertsen and Holman 41 demonstrated the presence of a wide range of hydrocarbons in herring (unspecified species) containing less than 0.1 ~ of this lipid class (Table 1). These workers detected 21 straight- and 8 branched-chain compounds. Of the straight-chain paraffins, those containing an odd number of carbon atoms predominated. The branched-chain compounds were primarily pristane and squalene.

It is reasonable to assume that squalene is biosynthesized in fish via the familiar pathways found in land animals and plants, i.e., through mevalonic

339

340 P R O G R E S S IN THE C H E M I S T R Y OF t A T S A N D O T H E R L I P I D S

TABLE 1.

Hydrocarbons occurring in Herring Oil as determined by Gas Chromatography

Straight chain components

Carbon % area number

14.0 0.3 15.0 7.5 16.0 0.6 17.0 9.0 17.6 2.2 18.0 1.7 19.0 10.5 20.0 0.3 21.0 6.0 22.0 0.3 23.0 0.8 24.0 0.3 25.0 9.5 26.0 trace 27.0 33.0 28.0 trace 29.0 9.0 30.0 trace 31.0 ~7 32.0 [ (trace) 33.0 l

I ,

t Flat and broad peak, From

Branched chain components

Carbon number

15.4

168 (pristane)

17.9

19.0

19.9

"~21t

22.2

26.3 (squalene)

the work of Lambertson and Holman. 41

% area

3.5

70.0

5.0

0.5

1.0

3.5

3.5

13.0

acid and farnesyl pyrophosphate intermediates 87 (see Vol. X, pp. 151-238). Furthermore, squalene is probably an important precursor in the formation of marine sterols, zl A purely biochemical role for squalene, however, does not provide a plausible explanation for the deposition of more than small percentages of this hydrocarbon. The inordinately large proportions of squalene in certain species of Squalidae 2s must serve a purpose that is not strictly biochemical. One can only speculate on the significance of high percentages of squalene and our further understanding of the matter is hampered by the fact that it is not known what proportion is derived via biosynthesis or absorbed through the intestine. We wish to suggest the possibility that high squalene content may serve a distinct function in certain fish, such as the control of the organism's density.

Avigan and Blumer 1 demonstrated a conversion of phytol to pristane in zooplanktonic copepods (two species of Calanus). The various calanids accumulate large amounts of pristane, particularly during the Spring planktonic

THE L I P I D B I O C H E M I S T R Y OF M A R I N E O R G A N I S M S 341

bloom. Because these copepods form an important link in the marine food chain, the pristane in these organisms is probably a major source of this hydrocarbon in higher forms of marine life. Furthermore, Avigan and Blumer 1 have speculated that this synthesis may be the most significant source of pristane in animal tissues and geological deposits. Nevertheless, it is not clear what portion of the large amounts of pristane found in some marine species is the result of diet or metabolism. With respect to both pristane and squalene an important question remains unanswered: "Do certain organisms scavenge these hydrocarbons from the environment to maintain their own life functions 2"

Lambertsen and Holman's report 41 showing that the herring deposits hydrocarbons resembling the alkyl chains of fatty acids suggests that fish possess the capability of converting fatty acids to hydrocarbons. The possibility that hydrocarbons are synthesized, at least in part, by intestinal microorganisms, as suggested by Bottino and Brenner, 6 implies that more than one major route might exist.

B. Glyceryl ethers 1. Composition and structure

It is not our intention to present an extensive discussion of the biochemistry of glyceryl ethers because this matter has been discussed in the chapter by Snyder a~ in the present series. We will, however, review the current literature pertaining to marine organisms.

As a general reminder, the glyceryl ethers studied so far have the ether linkage in position-1 of glycerol, each structure belongs to the D series, and the alk-l'-enyl glyceryl ethers (a, fl-vinyl ethers) have the cis configuration/f

The diacyl alkyl glyceryl ethers ("alkoxydiglycerides") represent a high proportion of the lipids of certain species. For example, in dogfish (Squalus acanthias) 5s and ratfish (Hydrolagus colliei) 7s these compounds occur as greater than 50 mole % of the total lipids. Wardle and Malins 8a have demonstrated the presence of 20 to 30 mole % of these compounds in the livers of several species of sharks caught at 800 meters in the South Atlantic (Fig. 1). In one species (Centroscynnus coelolepsis) diacyl alkyl glyceryl ethers appeared to be the only neutral glycerolipids present. The reader will note from an examination of Fig. 1 that wax esters predominated in each species.

Several studies have shown that small percentages of monoacyl alkyl glyceryl ethers occur in Squalus acanthias (< 1 mole % of the total lipids) 5~ and trace amounts have been detected in the starfish (Asterias forbesi). 2o, ~,5 These compounds appear to accompany high percentages of alkoxydiglycerides in marine organisms. Trace amounts of non-esterified alkyl glyceryl ethers have been detected in the livers of Squalus acanthias. 5~

t For convenience, the term "glyceryl ether" will refer to both the alkoxy compounds (e.g. chimyl alcohol) and the alk-l'-enyl glyceryl ethers (~, fl vinyl ethers). The term "alkyl" will be used to designate compounds having the alkoxy structure.

3 4 2 PROGRESS IN THE CHEMISTRY OF FATS AND OTHER LIPIDS

Current findings suggest that alkyl ethers of the phosphatide type, such as the analogs of phosphatidyl ethanolamine and phosphatidyl choline, are present in marine organisms. 19. 4s, s6 It is of interest that Lewis 45 reports the occurrence in squid (Loligo species) of alkyl ether analogues of phosphatidic acid. In fact, alkyl ether phosphatides containing an amino base were not detected.

Neutral alk-l'-enyl glyceryl ethers have been reported in several species of marine organisms. Diacyl alk-l'-enyl g!yceryl ethers have been found in starfish (Asterias forbesi) diverticulum (0.3 mole % of total lipid), z5 the liver of the dogfish (SquaIus acanthias) (0.2 mole % of total lipid), 52 and in the ratfish (Hydrolagus colliei) (about 5 mole % of total lipid)3 s

Small percentages of the monoacyl alk-l'-enyl glyceryl ethers have been found in the starfish (Asteriasforbesi) ( < 0.2 mole % of total lipid) 25 and in the hepatic lipids of Squalus acanthias ( < 0.1 mole % of total lipid). 52 Nonesterified /dk-l'-enyl glyceryl ethers are present as minor components ( < 0.1 mole % of total lipid) in the hepatic lipids of Squalus acanthias. 52 It is likely that these compounds accompany higher percentages of monoacyl and diacyl alk-l'-enyl glyceryl ethers in other marine organisms. Phosphorylated alk-l'-enyl glyceryl ethers have been detected recently in trace amounts in the hepatic lipids of Squalus acanthias ( < 0.1 mole % of total lipid) 5z and in the diverticulum of Asteriasforbesi. TM These compounds are also present in certain molluscs, s~

2. Cellular distribution The information available on the distribution of various lipid classes in

organs and subcellular fractions of marine species is sparse. The distribution of the glyceryl ethers, which are by no means the major constituents of most marine organisms, has been given preferential treatment. For example, Karnovsky et al.a~ conducted a chemical and histological study on the lipids of the pyloric cecum of Asteriasforbesi. Table 2 shows the compoklt!~ ~ of the

TABLE 2.

Analysis of Lipid Extracted from Various Cellular Fractions of Asterias forbesi. (The values are percentages of the total lipid of eacti cellular fraction.)

Lipid I II III IV "Droplets . . . . Nuclei . . . . Mitochondrla . . . . Microsomes"

Phosphorus Acctal l ipidst Unsaponiflable fraction Sterols+* Glyceryl ethers §

0.06 2.2

13.8 1.l 4.2

1.02 7.7

30.5 6.1 2.2

0.99 6.6

17.5 4.5 2.5

0.75 5.6

44.7 4.0 3.0

t Calculated as the acetal phosphatide of Thannhauser, Boncoddo, and Schmidt. s3 Calculated as Cholesterol.

§ Calculated as batyl alcohol. From the work of Karnovsky and coworkers. 37

FIG. 1. Thin-layer chromatogram of deep sea sharks: (1) Centroscynnus coelolepis, (2) Centrophorus, species unknown, (3) Deania, species unknown, and (4) Etnoprerus spinax. Silica Gel G developed with petroleum ether(3tHSO”C)-diethyl ether-acetic acid 90:10:1, v/v. Plate sprayed with chromic sulphuric acid. Major components from top to bottom: wax esters, diacyl alkyl glyceryl ethers, and triglycerides. (From the

work of Wardle and Malinsss)

FL-f.p. 342

THE LIVID BIOCHEMISTRY OF MARINE ORGANISMS 343

major lipid classes isolated via centrifugation in sucrose: Free lipid ("droplets") (I), "nuclei" (lI), "mitochondria" (III), and "microsomes" (IV) were separated. Aldehydogenic lipidg ("acetals") appear in higher proportions than alkyl glyceryl ethers in fractions II, III; and IV. In the free lipid, however, the alkyl glyceryl ethers clearly predominate over the aldehydogenic lipids. The high proportions of the aldehydogenic lipids in the organelles tends to implicate these compounds as serving an important function in cellular architecture. The role of alkyl glyceryl ethers in fat storage cells (I) cannot be explained at present.

Studies comparable tO those reported by Karnovsky et al. 37 have been conducted by Malins 5a Ori the hepatic lipids of Squalus acanthias. After intrahepatic injection of (1-14C):palmitic acid, the following fractions were separated by centrifugation in sucrose75: free lipid ("droplets") and total cellular debris (Table 3). No evidence was found for more than trace amounts of glyceryl ether phosphatides in either fraction. Reminiscent of the results of KarnovSky et al., 37 the alk-l'-enyl glyceryl ether phosphatides showed a clear preference for the merhbranous fraction. The free lipid contained the greater proportion of

TABLE 3.

Incorporation of Radioactivity into Droplet (Free Oil) and Membrane Lipids after Administration of (l-14C)-Palmitic Acid (6 hr after intrahepatic injection)

Lipid class

Triglyeerides Diglycerides Monoglycerides Free fatty acids

Sp: a (cpmh~m) Radioactivity (cpm)

Drop.?

Phospholipids aldehydes

Alkyl glyceryl ethers Diacyl

alkoxy chain Monoacyl

alkoxy chain Non-esterified

alkoxy chain AIk-l'-enyl glyceryl ethers

Diacyl aldehydes

Monoacyl aldehydes

Non-esterified aldehydes

Drop. Mem.

I 354 2100 5590 22,750 1295 1172

16,500 44,400

63 6

1293 135

3,000,000 89,600 13,800

17,500,000

390 73

4631 555 263 282

1095 17,700

288 390,000 <4 34,500

65~ - - 6 ~

~37 2175

- - 5000 504 90O - - 74,000 621 8600 770 225 828 225

Mere.++

146,000 275,000

22 1,780,000

158;000 7300

7OO0

130 130

70

3OO0 44 44

? Radioactivity of total iipids recovered was 2.3 × 107 cpm (14.9 g). ++ Radioactivity of total lipids recovered was 2.38 × i06 cpm (218 rag). From the work of Malins. 5e

344 P R O G R E S S IN THE C H E M I S T R Y OF FATS AND OTHER L I P I D S

the diacyl alkyl and alk-l'-enyl glyceryl ethers. However, the nonesterified and monoacyl alk-l'-enyl glyceryl ethers showed a significant preference for the membranous fraction, which suggests their active involvement in metabolism. The data on the incorporation into ether lipids of radioactivity from the labeled palmitic acid will be discussed later in this section (3a and 3b).

Figure 2 shows a wax embedded liver of Squalus acanthias. It is readily apparent that a major portion of the hepatic lipids are stored intracellularly in lipid vacuoles. Other species of sharks having fatty livers, such as Scyliorhinus caniculus, show similar patterns. The recent work of Denton and MarshalP 6 suggests that fatty deposits in fish are fundamentally related to the fine control of density. It is reasonable to assume, therefore, that the composition of the fat within the vacuole is related to its function as a hydrostatic organ. An important biochemical role for glyceryl ethers in the fat vacuole is suggested by their occurrence in high percentages aT, 56 and their active turnover. 19, 52

3. Metabolism In the last few years, studies with carbon-14 and tritium have begun to

elucidate the pathways by which the glyceryl ethers are metabolized. Clearly, as suggested by Snyder's review, s~ our understanding of the pathways involved is only embryonic. The contributions to glyceryl ether metabolism that can be gleaned from the marineworld are well worthy of discussion.

(a) Incorporation of labeled precursors. Ellingboe and Karnovsky 19 studied the incorporation of carbon-14 and tritium into the glyceryl ethers by incubating digestive glands of ,4sterias forbesi with solutions of (1-14C)-acetate, (1-14C)- stearic acid, (1-14C)-stearaldehyde, (l-14C-l-aH)-stearaldehyde, and (1-1ac-I,I'- aH)-stearyl alcohol. The labeled acetate was readily incorporated into neutral and phosphatide alkyl glyceryl ethers, but only slightly into the alk-l'-enyl structures. Both (l-14C)-stearic acid and (1-14C)-stearaldehyde were incorporated into alkyl ethers. Proportionately more incorporated radioactivity, however, resulted from the stearaldehyde. Isotope ratios and relative specific activities indicated that (1-~4C-l,l'-aH)-stearyl alcohol was a better precursor than the (l-14C-l,l'-aH)-stearyl aldehyde in the synthesis of the alk-l'-enyl glyceryl ethers. However, the fatty alcohol was more favorably incorporated into the alkyl glyceryl ethers.

Malins, ~2 using Squalus acanthias, administered (l-14C)-palmitic acid via intrahepatic injection and then examined the incorporation of radioactivity into a variety of alkyl and alk-l'-enyl glyceryl ethers of the liver. A reductive incorporation into alkyl and alk-l'-enyl ether chains was demonstrated. Considerably lower specific activities were found in the diacyl alk-l'-enyl glyceryl ethers and diacyl alkyl glyceryl ethers in comparison to both types of nonesterified and monoacyl derivatives.

Friedberg and Greene, 2a working with the isolated stomachs of Squalus acanthias and the skate (2~aja erinacea), found that carbon-14 labeled fatty alcohols and acetate, but not labeled fatty acid, were readily incorporated into alkyl glyceryl ethers. In contrast, the in vivo studies of Malins 5~ demonstrated

THE LIPID BIOCHEMISTRY OF MARINE ORGANISMS 345

tha t only a sl ight a m o u n t o f rad ioac t iv i ty , in compar i son to the alkyl and a l k - l ' - e n y l glyceryl ethers, was inco rpora t ed into fat ty a lcohols o f the liver

after the admin i s t r a t ion o f ( l-14C)-palmitic acid. MalinsS2, s4 has shown tha t chimyl-( l -14C)-alcohol is rap id ly metabol ized in

the liver o f Squalus acanthias. A significant po r t ion o f incorpora ted rad ioac t iv i ty was found in bo th the neutra l and phospha t ide a lkyl and a l k - l ' - e n y l glyceryl ethers. However , the greatest a m o u n t o f rad ioac t iv i ty was detected in the free and esterified fat ty acids (Table 4).

(b) Interconversions. The acetate exper iments o f El l ingboe and K a r n o v s k y 19 suggest tha t a convers ion f rom a l k - l ' - e n y l glyceryl e ther to alkyl glyceryl

TABLE 4.

Incorporation of Radioactivity into Lipids after Administration of Chimyl-(1-14C)-Alcoholt

Lipid class

Triglycerides Diacyl glyceryl ethers

Alkoxy chain Diacyl alk-l-enyl

ethers Aldehydes

Diglycerides + monoacyl alkyl glyceryl e thers

fatty acids Monoacyl alkyl

glyceryl ethers Alkoxy chain

Monoacyl alk-l'-enyl glyceryl ethers

Aldehydes Monoglycerides Non-esterified alkyl

glyceryl ethers AIkoxy chain

Non-esterified alk-l '- enyl glyceryl ethers

Aldehydes Phospholipids

Aldehydes Alkoxy chain

Fatty acids

Time in hr

4'0 10.5

% of injected dose

(10-a)

m

mole % cpmh~m

5.2 1.2

5.7 0.24

0.28 5 × l0 -a 2.8 0.04

49.3 0.12

0.02 1 × 10 -4

0.82 0.23

160 9.9

0.3

?6

5.0

288

!74

?l

% of mole % injected dose

(10 -a)

170 84.0 4.25 11 4 1.4 I0.5

0.30 4 × l0 -a 0.04 4 x 10 -a

6.28 0.12

42.5 0.36

0.73 1 × 10 -a 0.47 1 × 10 -a

20.7 0.11

820 0.13

0.77 2 x 10 -4 140 2.5

1.9 0.02 32.5 0.47

270 2.8

cpm/g.m (I03)

0.2 0.04 0.01

6.6 0.9

4.6

10.4

50.8 30.0 18.9

6× l0 -~

m

270 4.8 6.51 6.0

84.0

? In the 4.0 hr and 10.5 hr experiments, I01 ~,c and 81 ~c were administered, respectively. From the work of Malins. 52

346 P R O G R E S S IN THE C H E M I S T R Y OF FATS AND O T H E R L I P I D S

ether is unlikely in Asteriasforbesi because of the lower specific activities of the former compounds at all time periods. In addition, the lower specific activities of the diacyl alkyl glyceryl ethers in comparison to the alkyl ether phosphatides does not lend support to a proposal by Thompson 85 that the diacyl alkyl glyceryl ethers might be precursors of alkyl and alk-l'-enyl ether phosphatides. The very low specific activities found in the diacyl alkyl glyceryl ethers by Malins,52, 54 after the administration of both (l-x4C)-palmitic acid and chimyl- (l-14C)-alcohol, suggest that in Squalus acanthias the diacyl .alkyl glyceryl ethers represent a "terminal point" in ether lipid biosynthesis. This conclusion is supported by the evidence obtained from the cell fractionation studies with Squalus acanthias56 in which the radioactivity of the intracellular lipid was compared with that obtained for total cellular debris. The diacyl alkyl glyceryl ethers isolated from each fraction had extremely low specific activities in comparison to other ether structures (Table 3). It seems unlikely that a discrete pool of diacyl alkyl glyceryl ethers is present that might serve as active intermediates in the overall biosynthesis of either alkyl or alk-l'-enyl glyceryl ethers in this species. Nevertheless, the extensive oxidative cleavage of the large amounts of diacyl alkyl glyceryl ethers in the liver indicates that these compounds have an active metabolism which might be related in a fundamental way to the function of the liver as a hydrostatic organ.16, 5~

Ellingboe and Karnovsky 19 report the isolation of "unknown compounds" from the neutral and phospholipid fractions of Asterias forbesi. After the administration of double labeled aldehyde, a high degree of labeling and retention of tritium, relative to carbon-14, was found with these compounds. These findings evoked the hypothesis that the unknown compounds are involved as immediate precursors of alkyl glyceryl ethers.

Cyclic acetals were detected in the studies with double labeled aldehyde 19 and these compounds, present in small amounts, incorporated significant radioactivity. The retention of tritium, relative to carbon-14 in the cyclic acetals suggests the possibility that these compounds are involved in pathways leading to the biosynthesis of alk-1 '-enyl glyceryl ethers. The cyclic acetals are worthy of further study, notably because of their occurrence in high percentages in certain species, such as the sea anemone (Anthropleura elegantissima). 3

The possible involvement of fatty alcohols in alk-l'-enyl glyceryl ether biosynthesis was suggested by the work of Keenan, Brown, and Marks sa with the dog heart-lung machine. Research has now been instigated on several marine species that allows this possibility to be investigated further. Ellingboe and Karnovsky 19 tested the hypothesis that fatty alcohols might be directly incorporated into alk-l'-enyl ether chains of glyceryl ethers in their studies of Asterias forbesi. The evidence did not support such a precursor role. However, the isotope ratios and relative specific activities from these experiments led them to believe that fatty alcohols and fatty aldehydes could be converted to alkyl and alk, l'-enyl glyceryl ethers, respectively, without an intermediate oxidation step. Friedberg and Greene 2a also concluded,that alkyl glyceryl ethers can be synthesized directly from fatty alcohols in their studies with Raja erinacea


and Squalus acanthias. However, the experiments conducted by Malin s~5 in vivo with Squalus acanthias, using (l-14C)-palmitic acid, demonstrated that the specific activities and total radioactivities of free and esterified fatty alcohols, over several time periods, were very much lower than the same values for a variety of alkyl and alk-l'enyl glyceryl ethers. Perhaps the discrepancies between the work of Malins 55 and Ellingboe and Karnovsky 19 and Friedberg and Greene ~ are due to the different experimental conditions and the fact that different organs were studied.

It has been suggested that the diacyl phosphatides might be precursors of glyceryl ethers.S5 The (1-14C)-acetate experiments of El!ingboe and Karnovsky 19 with Asterias forbesi clearly demonstrated a high rate of incorporation into diacyl phosphatides. Similar results were obtained by Malins 5~ with (1-~4C) - palmitic acid in studies of glyceryl ethers in Squalus acanthias liver. The pathways in a conversion of diacyl phosphatides to alkyl glyceryl ethers would be expected to involve the formation of alkyl ether phosphatides at an early stage of biosynthesis. Malins, 5~ however, was unable to detect more than possible traces of such compounds. The alk-l'-enyl glyceryl ether phosphatides were present, however, to the extent of about 0.1 mole % of the total lipids. Small percentages of the alkyl glyceryl ethers were isolated from a phospholipid fraction of the liver after the administration of chimyl-(IJ4C)-alcohol via intrahepatic injection. The specific activities of these glyceryl ethers were high, but the values were considerably lower than those for certain neutral ethers, such as the monoacyl glyceryl ethers. A capacity to phosphorylate alkyl glyceryl ethers was demonstrated by the chimyl alcohol experiments, but it is not known if such a conversion is important in glyceryl ether metabolism.

ThompsonS4, s5 has presented rather convincing evidence that the alk-l'-enyl glyceryl ethers are derived from alkyl glyceryl ethers in the terrestrial slug (Arion ater). Supportive evidence for such a conversion in marine species was brought forward by Malins 52, 54 in his studies with labeled chimyl alcohol administered to the dogfish. In two experiments, a 2:1 ratio of specific activities of nonesterified alkyl glyceryl ethers to the analogous nonesterified alk-l'-enyl glyceryl ethers was observed (Table 4). This, and other evidence, suggested that the chimyl alchol was converted directly to the analogous alk-l'-enyl glyceryl ether, probably via biodehydrogenation. However, the results of Ellingboe and Karnovsky 19 with the starfish do not militate in favor of a conversion from alkyl to alk-l'-enyl glyceryl ethers. Notably, the evidence does not support the role of alk-l'-enyl glyceryl ethers as immediate precursors of alkyl glyceryl ethers in marine organisms. 19, 52

(c) Oxidative cleavage of ether bonds. It is now well established that enzyme systems capable of oxidatively cleaving glyceryl ethers are present in a wide variety of terrestrial and aquatic animals, sz It has been reported by Malins, Wekell, and Houle 59 that the trout (Salmo gairdneri) is able to extensively oxidize in the intestine alkyl glyceryl ethers obtained from a diet rich in these compounds so that only trace amounts are deposited in its tissues. The studies of Lewis, 45 with several marine species, evoked the hypothesis that elasmobraneh fish,

348 P R O G R E S S IN THE C H E M I S T R Y OF FATS A N D O T H E R L I P I D S

which contain large proportions of alkyl glyceryl ethers, may not have developed enzyme systems capable of oxidatively cleaving the ether linkage. This concept was supported by the experiments of Pfleger, Piantadosi, and Snyder 7o and of Thompson s4 with other organisms. The extensive oxidative cleavage of chimyl- (l-14C)-alcohol reported by Malins 5~ in Squalus acanthias liver, however, suggests that the hypothesis proposing an inverse relationship between alkyl glyceryl ether content and activity of ether cleaving enzymes is not tenable for all species. In fact the evidence now strongly supports the concept that alkyl glyceryl ethers, although present in high percentages, are oxidized and re- synthesized rapidly in the liver of Squalus acanthias.

The observation has been made by Wekeli and Wekel191 that enzyme preparations derived from trout (Salmo gairdneri) intestine appear to be quite stable during isolation and storage. Salmo gairdneri, a readily available species, might therefore serve as a good source of purified enzyme fractions for future studies on oxidative cleavage of the ether bonds of alkyl glyceryl ethers.

C. Wax esters

1. Composition and structure

Various proportions of wax esters are present in the lipids of a variety of marine species. 2, 24, 27, 29, 34, 6,~, 64, 65 It has been shown, however, that very large proportions of these compounds are present in certain species. Nevenzel, Rodegker, and Mead 65 report that the lipids of 2~uvettus pretiosus muscle are comprised of 92 9/00 wax esters, whereas the liver contains less than 4 ~. Nevenzel et aL 64 dissected various parts of a coelacanth (Latimeria chalumnae) and found very high percentages of wax esters in the lipids of each sample. The "swim bladder" contained the highest percentage (97~o) and the smallest amount was detected in the hepatic lipids (less than 8 ~o). Iyengar and Schlenk 34 reported that the roe of the mullet (Mugil cephaha.) contained 67 ~ wax esters. Also, significant proportions of odd-chain fatty acids and alcohols are present. Wardle and Malins ss have demonstrated the presence of high proportions of wax esters in the liver of several species of deep sea sharks (Fig. 1). Also, Nevenzel and Kayama 63 have detected high proportions (,~ 90 ~) in the lipids of the lantern fish (family Myctophidae).

2. Metabolism Although certain marine organisms are known to synthesize large amounts of

wax esters, only a few studies of metabolism have been undertaken. 24, 55, 63 Nevenzel and Kayama 63 worked with midwater lantern fish (family Myctophidae) which contain wax esters ranging from Cao to C38 chain length. In one to three hours after intramuscular injection, carbon-14 radioactivity from labeled acetate, 1-hexadecanol, palmitic acid, and oleic acid was incorporated into wax esters to the extent of I to 5 ~o. A reductive incorporation into alcohol was observed. This investigation suggested that the de novo synthesis of fatty acids


and long-chain alcohols takes place readily. It was also shown that long-chain alcohols are preferentially incorporated into wax esters over glycerolipids.

The studies in vivo of Malins 55 with Squalus acanthias revealed that radioactivity of administered (l-14C)-palmitic acid is only very slightly incorporated into wax esters. Also, only a slight radioactivity was associated with free fatty alcohols. The studies of Friedberg and Greene 24 with Squalus acanthias liver indicated that the microsomal and supernatent fractions were active in the synthesis of wax esters. The synthesis was promoted by the addition of emulsi- fiers, such as Triton X-100, Tween 20, and bile salts. Perhaps the most interesting fnding was that wax synthesis occurred without the intermediate acyl CoA derivative. Also, added cofactors (AT?, CoA, Mg ++, CTP) did not influence the rate or extent of the conversion and diisopropylfluorophosphate inhibited the reaction. It is an intriguing possibility that the lack of a requirement for ATP might reflect a lessening of the demand on the respiratory system in a fish that is known to migrate in water very low in oxygen.

It has been suggested that the synthesis of wax esters in Squalus acanthias takes place under conditions where water is essentially excluded. This reaction would then be akin to the esterification of cholesterol by pancreatic cholesterol esterase in terrestrial animals eg.

Wax esters may be involved in the control of the hydrostatic properties of fish undergoing vertical migrations, as suggested by the high percentage of these compounds in certain deep sea sharks a8 and the lantern fish. 6a It is a tempting hypothesis that pressure (depth), and possibly temperature or oxygen concentration, may play a significant role in regulating the metabolism of wax esters. At present, however, the influence of such parameters has not been demonstrated.

D. Triglycerides

The triglyceride fatty acids of marine organisms are usually characterized by relatively high percentages of polyenoic acids, notably eicosapentaenoic acid (20:5), docosapentaenoic acid (22:5) and docosahexaenoic acid (22:6). 2e Exceptions to this general rule are some species of sharks. For example, the liver of the dogfish (Squalus acanthias) contains large proportions of monoenoic acids of the C20 and Cz2 series and relatively small percentages of the characteristic polyenoic acids. 57. 58 The triglycerides of the mullet (Mugil cephalus) have significant amounts of odd- and branched-chain acids. 4a, 79 Furthermore, some cetaceans, such as the porpoise (family Delphinidae), are characterized by triglycerides esterified with high percentages of iso-valeric acid. 30 Of the thousands of marine species, only a few have been examined thus far by modern techniques, such as gas-liquid chromatography, so only tentative conclusions can be drawn.

1. Positional distribution of fatty acids In the last few years notable advances have been made in techniques for the

analysis of glyceride structure. With the advent of "stereospecific analysis ''9 it

350 PROGRESS IN THE CHEMISTRY OF FATS AND OTHER LIPIDS

is now possible to examine the fatty acids on each of the 3-positions of glycerol. These techniques, dev¢10ped skilfully by Hans Brockerhoff, have been employed for the analysis of a variety of marine fish, mammals, and invertebrates. 11 Certain general rules can now be formulated about the positional distribution of the fatty acids in triglycerides of marine species. It must be understood that general rules outlined below, based on analyses of a limited number of species, represent tendencies rather than structures.

(a) Marine fish and invertebrates. In marine fish, positions-1 and 3 attract the saturated and long-chain acids (Table 5). Position-3, however, shows a preference for the longer-chain compounds, such as docosenoic acid (22:1). Position-2 shows a clear preference for the characteristic polyunsaturated acids, eicosapentaenoic acid (20:5), docosapentaenoic acid (22:5) and docosahexaenoic acid (22:6). The triglycerides of most fish also appear to show a preference for palmitic acid (16:0) in position-2. The acids esterfied to glycerol at the central position constitute a stable "2-monoglyceride backbone" that is carried through the food chain ~4 as will be discussed later. Analyses from the few marine invertebrates examined suggests that the general rules for marine fish might also apply (Table 5). The scallop exhibits a slight deviation, however, in that there is no clear preference for the deposition of long-chain acids on position-3:1

It will be suggested later that the acylation of the exogenous 2-monoglyceride backbone is probably a major route by which marine triglycerides are biosynthesized. Nevertheless, consideration should be given to certain other pathways common to terrestrial species. For example, in the metabolism of lyso- phosphatidyl choline and lyso-phosphatidyl ethanolamine in vitro, saturated acids are preferentially esterified at position-1 and unsaturated and short-chain acids at position-2, s, 4~ The diacyl phosphatides so formed could then yield triglycerides of the characteristic type via dephosphorylation and acylation of the resulting 1,2-diglycerides. Furthermore, the tendency to accumulate polyunsaturated and short-chain fatty acids in position-2 can be rationalized in terms of the specific properties of the substrate. The fatty acid pattern of position-2 in fish is said to be reminiscent of a "reverse-phase" chromatogram, a That is to say, part of the molecule is more attracted to a hydrophilic phase than to a lipophilic phase. The acylation of 1,2-diglycerides, for example, is presumed to take place at lipid-water interfaces, so the orientation of the molecules between these two phases might well be influential in determining whether acylation occurs from primarily a lipophilic or a hydrophilic pool of fatty acids. The generally low solubility of 1,2-diglycerides in polar phases might determine, at least in part, the preference shown for long-chain acids, such as docosenoic acid (22:1), for position-3 of triglycerides.

(b) Marine mammals. Marine mammals offer a somewhat different picture from marine fish and invertebrates (Table 5). 11 In the few species examined, the 20:5, 22:5, and 22"6 acids are preferentially esterified at positions,1 and 3. The seal and polar bear, however, show a similar pattern to marine fish and invertebrates in that the shorter-chain acids, such as myristic acid, accumulate

÷.l

-.t.

pr

Nu*

~

÷÷~÷ A

AAA

~"

0

~ ~

~ o

~ M

o


on position-2. Furthermore, the sei and pilot whales show some tendency to deposit polyenoic acids on position-2.

The importance of diet on glyceride composition and structure is evident with most marine animals studied. Nevertheless, as previously mentioned, the porpoise and some species of whales contain high percentages of iso-valeric acid in the depot fats. Iso-valeric acid is not derived directly from a marine diet, but is presumably a product of leucine metabolism. Studies in our laboratories 51 indicate that the porpoise (family Delphinidae) stores high percentages of iso-valeric acid and other short-chain acids as triglycerides. It is of interest to note, from a comparative biochemical point of view, that land mammals appear to have a very low tolerance for iso-valeric acid as evidenced by the terminal disease in human infants, iso valericacidemia. 66

2. Enzyme specificity

Litchfield 47 has observed that the positional distribution of the 22:5 and 22:6 acids in triglycerides of several species of marine animals can be predicted from a simple proportionality equation of the type y ---- kx. The proportionality constant (k) probably reflects an enzyme specificity and the factor x the concentration of the 22:5 and 22:6 acids available for triglyceride synthesis. Equa- tions of this type might serve a useful purpose in estimating the composition of triglycerides of natural fats.

Bottino, Vandenburg, and Reiser 7 have shown that the 20:5 and 22:6 acids located on the 1,3-positions of whale oil triglycerides are resistant to hydrolysis in vitro with hog lipase. However, the accompanying 22:5 acids are hydrolyzed without difficulty. The resistance of the 20:5 and 22:6 acids might be due to the close proximity of the first double bond to the carboxyl group in these structures. This work raises the interesting question whether these results can be duplicated with lipases obtained from marine mammals.

The results of Bottino, Vandenburg, and Reiser 7 clearly emphasize the need for a better understanding of the glyceride structures of marine organisms. Unfortunately, only a paucity of information exists on specifc types o f triglycerides. The effect of the whole structures of individual glycerides on the resistance to lipase hydrolysis remains an unanswered question. Perhaps the resistance is primarily related to certain glyceride types, rather than to the unique structure of individual fatty acids. The complexities involved in the analysis of specific glycerides is well documented by the study of sable fish (Anaplo fimbria) by Dolev and Olcott. is

The rules formulated so far on the positional distribution of fatty acids on the triglycerides of marine animals must be considered as tentative. The marine environment comprises a vast number Of species, so that rules formulated today will undoubtedly undergo modification over the years as new samples are examined. In the process, a deeper understanding of the glyceride patterns will evolve.


E. Phospholipids 1. Composition and structure

A spectrum of phospholipids comparable to that of land animals is present in marine organisms. 5°, 53, 81 For example, compounds such as lecithin, phosphatidyl ethanolamine, cardiolipin, inositol phosphatides, cerebrosides, sphingo- myelins, lyso-lecithins, and phosphatide plasmalogens are reported to be present in the South African pilchard (Sardinia ocellata). 81 Marine life, however, has yielded a number of unusual phospholipids that are interesting in terms of comparative biochemistry. Bergmann and Landowne 8 have reported the presence of large percentages of acetal phosphatides in the sea anemone (Anthropleura elegantissima). The possible uniqueness of this finding was suggested later by the work of Rapport and Alonzo 73 with several species of sea anemone other than Anthropleura elegantissima. These workers reported that the aldehydogenic phospholipids were primarily alk-l'-enyl ether structures. The probability that acetal structures are not rare in the marine biosphere was revived recently when Ellingboe and Karnovsky 19 detected cyclic acetals in the starfish ( Asterias forbesi ).

Landowne and Bergmann 42 found an unusual sphingolipid in Anthropleura elegantissima that differed from spingomyelin in that the hydroxyl group on the 3-carbon of sphingosine was acylated. Choline was not detected and the phosphatide group was present as a monoester. The unusual composition of Anthro- pleura elegantissima has also been demonstrated by Rouser and coworkers 77 who detected the presence of ceramideaminethylphosphonate in this species. The work of Quinn 72 and Hori 8z suggests that compounds having a carbon-to- phosphorus bond occur in a variety of marine invertebrates. The enticing possibility that marine life offers a wide spectrum of unique phospholipids is also suggested by the work of Nakazawa 62 and Igarashi, Zama, and Katada. aa Nakazawa detected an unusual glycolipid in an oyster and Igarashi found a threonine containing lipid in the tunny (Thynnus orientalis).

Two important studies have been undertaken in the last decade on the fatty acids esterifying positions 1 and 2 of glycerophosphatides. Brockerhoff, Ackman and Hoyle 13 determined the positional distribution of the acids in the lecithins and phosphatidylethanolamines of cod (Gadus callarias), lobster (Homarus americanus), and scallop (Platopecten magellanicus). Menzel and Olcott el analyzed the lecithins of tuna (Thunnusalalunga), menhaden (Brevoortia tyrannus), and salmon (Oncorhynchus tshawytscha) muscle. Table 6 shows the distribution patterns of the fatty acids between positions 1 and 2. It is evident that the characteristic polyunsaturated acids (20:5 and 22:6) are found preferentially esterifying position 2, whereas the saturated acids favor position-1. The work of Menzel and Olcott ~1 and of Brockerhoff, Ackman, and Hoyle 13 suggests that no distinct tendency exists for short-chain acids, such as myristic acid (14:0), to preferentially esterify position-2, as is evident with the triglycerides of fish.

Present evidence clearly suggests a preference for polyunsaturated acids


TABLE 6.

Specific Distribution of the Principle Fatty Acid from the Isolated Lecithins of Marine Fish

Identification is based on relative retention times of authentic standards of the fatty acid methyl ester or on the relative retention times of homolOgs followed by hydrogenation and rechromatography. The notation in the first column includes the number of carbon atoms followed by the number of double bonds. " t r " denotes

trace amounts; e.g., <0.1%

Weight ~ of specific fatty acid isolated from Tentative

identification Salmon Menhaden Tuna of fatty acids

Total 1 2 Total 1 2 Total 1 2

0.7 1.9 0.6 0.7 14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 20:4 20:5 22:1 22:4 22:5 22:6

22 37 10 8.0 ! .4 3.8 2.0 2.7 tr 6.7 8.2 23

tr tr tr tr tr tr

1.0 0.6 0.3 12 14 17 0 0 0

tr tr tr tr tr tr 43 33 46

0.5 tr 1.3 tr tr tr 45 78 6.2 tr 0 tr 3.2 5.9 0.9 7.4 2.3 13 1.0 tr 2.4

tr tr tr 2.2 tr 0.9

13 1.7 29 0 0 0

tr tr tr tr tr tr 28 12.5 42

tr 1.0 tr 1.8

19 36 5.0 2.1 tr 2.3 5.0 7.9 1.0 7.6 6.4 16 0.7 tr 2.2 0.8 tr !.1 2.7 1:3 5.0 7.1 8.4 15 0 0 0

tr 0 0.5 tr tr tr 55 39 48

From the work of Menzel and Olcott. sl

at posit ion-2 of mar ine phospholipids. However, in contrast to the triglycerides of fish, too few studies have been conducted to allow even tentative conclusions to be made on the dis t r ibut ion pat terns of other acids.

2. Metabolism

There has been a greater concern with the metabol ism of neutral lipids in mar ine organisms than with the phospholipids. Perhaps it is unfor tuna te in terms of comparat ive biochemistry that more effort has no t been directed toward the phosphol ipids in mar ine life. At the present time, our knowledge of phosphol ipid metabol ism is at best rud imentary and our present interest is still fixed at the structural level.

The structural investigations of Brockerhoff 8 have implied an impor tan t role for phosphol ipids in mar ine animals that is reminiscent of pathways described by Weiss and Kennedy sg, 90 and Lands and Har t 4a, 44 in which phos-

phatidic acids and a-glycerophosphate are impor tan t precursors of triclycerides.

THE LIP ID BIOCHEMISTRY OF MARINE ORGANISMS 355

The work of Porcellati et al. 71 offers some stimulation to further work in phospholipid metabolism of marine organisms. Their report on studies of the teleost, Aneiurus nebulosus, with labeled L-serine ethanolamine phosphate (I) and L-threonine ethanolamine Phosphate (II) was conducted on a microsomal fraction. The results indicated that enzymes were present capable of converting I and II to phospholipids more readily than vi~i a cytidine nucleofide pathway.S9,90

Modification in cellular structure is probably invo|~ed in adaption to the unique conditions of the marine environment, such as extreme pressure variations in vertical migrations. The intimate connection between phospholipids and cell structure suggests that this class of lipids might play a more distinct, perhaps more dramatic, role than in terrestrial animals. We are inclined to believe, for these reasons, that studies Of phospholipid metabolism in marine life offer some encouraging possibilities for future work.

F. Lipoproteins

The lipoproteins of fi.~h offer a stimulating area of research ifi comparative biochemistry and physiblogy. Unfortunately, however, the lipoproteins have not been studied too extensively.

Olley ea has shown that muscle phospholipids of various teleost and elasmo- branch fish are qualitatively similai'. Phosphatidyl choline and phosphatidyl- ethanolamine were the main constituents. Small proportions of phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerophosphatides, arid sphingo- myelin were detected. The blood lipoproteins of the lobster (Homarus americanus) have been studied by Bligh and Scott. 4 The results showed that phospholipids make up 65 % of the lipids iri the hemolymph. Phosphatidyl choline and phosphatidyl ethanolamine were the main constituents. The neutral lipids were primarily triglycerides and free ster01s, but no esterified sterols were detected. The compositional studies of Lauter, Brown, and Trams 40 have demonstrated that the very low density lipoproteins of Squalus acanthias carry the major proportions of the lipids: The free fatty acid content of the lipoprotein fractions is significantly higher than in comparable fractions from mammals. An exception is the d < 1.21 fraction where the lipids were in lower proportions. The results suggest the prob~ibility that circulating lipoproteins are derived from the intestinal tract. A fundamental role for the blood in fat transportation in Squalus acanthias is also indicated by the findings of these workers.

While on the subject of the lipoproteins, we are prompted to give consideration to the anatomy of Squalus acanthias with respect to fat transport. Because no thoracic duct exists, and a lymphatic system is not apparent on dissection, the inordinately large amounts of fat ingested are likely to be transported by the vascular system: If the greater portion of the ingested lipid, notably the long- chain components, is absorbed directly into the portal blood, the dogfish represents a clear exception to generally accepted concepts of fat absorption, s0

356 P R O G R E S S 1N THE C H E M I S T R Y OF FATS AND OTHER L I P I D S

Unfortunately, no work has been reported to shed light on this most intriguing possibility.

With respect to future studies, the role played by wax esters and glyceryl ethers in fractions, such as the blood lipoproteins, is of immediate interest in those species which deposit large proportions of these compounds in their tissues. Although it is well established that glyceryl ethers are present in high proportions in Squalis acanthias, 5s no mention was made of their occurrence in the lipoproteins by Lauter, Brown, and Trams 49 The role played by diacyl alkyl glyceryl ethers in the blood lipoproteins of this species is still to be elu- cidated.

I I I . E N V I R O N M E N T A L I N F L U E N C E S

A. Food chain and symbiosis

The influence of the food chain on lipid metabolism of marine organisms is well documented by the work of Brockerhoff, Hoyle, and Ronald 12 with rat, lobster, cod, and trout fed glyceryl-l,3-dioleate-2-palmitate-l-14C. The triglycerides from the liver and carcass of the rat showed no retention of the 2-monoglyceride structure beyond one day. In the fish and invertebrates, however, there was still retention after four weeks. It appeared, therefore, that the characteristic 2-monoglyceride structure might be carried through the food chain and possibly originate in the phytoplankton. Support for its origin at a primative level was brought forth when it was shown that phytoplankton exhibit the 2-monoglyceride structure 14 and that the digestive lipases of marine organisms, as exemplified by the skate (Rajidae family), are probably specific for positions-1 and 3 of triglycerides. 1° The role of the aquatic food chain in temperature induced alterations in fatty acid metabolism will be discussed in the section on Temperatures and Pressure (depth) (IIIB).

The importance of symbiotic relationships in the marine world and their relationship to the lipid composition and metabolism of various marine species was suggested by the work of Avigan and Blumer 1 with the copepods. For example, the occurrence of pristane in Calanus may be the result of associated microorganisms converting the ingested phytol, or phytanic acid, to pristane. The pristane might then be absorbed and stored in the host's tissues. Difficulties involved in further pursuing this hypothesis are related primarily to the fact that the use of antibiotics results in the death of both the microorganisms and the copepods. Another example of a symbiotic relationship influencing lipid biochemistry has been reported by Bottino and Brenner. e Their studies suggested that the branched-chain acids of fish are the result, at least in part, of synthesis by microorganisms of the intestine. Although the data to date is sparse, studies on the influences of the food chain and symbiosis on the biochemistry of marine organisms offer a refreshing and productive area for future studies.


B. Temperature and pressure (depth)

1. Temperature It is now well established that an inverse relationship exists between the

temperature of a tissue and the unsaturation of the tissue fatty acids. Un- fortunately, studies on the influence of temperature on lipid composition and metabolism have been conducted largely on total fatty acids without regard for the lipid classes involved. Nevertheless, the work reported so far ably documents the importance that must be attached to those biochemical changes that can be brought about in lipids by environmental factors.

Hoar and Cottle zl have shown that the lipid content of goldfsh (Carassius auratus) decreases with increasing environmental temperature and Roots and Prosser 76 have suggested that temperature acclimation involves a modification of the lipids of the nervous system to permit normal functioning at various temperatures. In a study by Lewis, 46 several marine ectotherms caught in Artic waters were found to have lower percentages of saturated fatty acids, notably stearic acid and palmitic acid, and higher proportions of palmitoleic acid than those species obtained from more temperate waters. Farkas and Herodek 22 have demonstrated that C20 and C2z polyunsaturated fatty acids of certain freshwater crustaceans increase with decreasing water temperature. In some species the proportions of polyunsaturated acids exceeded the values characteristic of marine fish. Kayama, Tsuchiya, and Mead aa fed brine shrimp to guppies (Lebistes reticulatus), kept at different temperatures, in a study of an aquatic food chain. The fish maintained in warmer water showed a relative decrease in 16:1, 18:1, and 22:6 acids. The studies of Johnson and Roots a6 with goldfish (Carassius auratus) also support the rule that higher temperatures result in the preferential deposition of fatty acids of lower unsaturation.

Knipprath and Mead 40 studied the influence of temperature on the fatty acid patterns of mosquitofish (Gambrusia affinis) and guppies (Lebistes reticulatus) (Table 7). In the mosquitofish study, one group was adapted to cold water (14 ° to 15°C) and another to warm water (26 ° to 27°C). Each group was maintained on a diet of brine shrimp. Characteristically, the lipids showed an increase in unsaturation in cold water. The major.changes observed at the lower temperature were a decrease in palmitic and stearic acids and an increase in docosahexaenoic acid. Unexpectedly, the arachidonic and linoleic acid levels decreased slightly at the lower temperature. The presence of only trace amounts of linolenic acid in the fish kept in cold water suggests that the source of the characteristic 22:6 acid is likely to be the diet. The 22:6 acid might result from elongation and desaturation of a 20:5 acid present in the brine shrimp, rather than from endogenous linolenic acid.

Two groups of guppies were studied at the same temperature levels (Table 7). One group was raised on a complete trout chow diet (group A) and another on ether-extracted trout chow (group B). Each group developed different fatty acid patterns. At the lower temperature, group A showed an increase in linoleic acid and a decline in arachidonic acid. In group B, however, these tendencies


TABLE 7.

Fatty Acid Compositiont off ish Maintained at Different Temperatures. Column 1." Mosquito Fish. Column 2: Guppies .Raised on Complete Trout Chow Diet.

Column 3: Guppies Raised on Ether-Extracted Trout Chow Diet

Fatty acid

14:0 14:1 15:0 i6:0 16:1 16:2 16:4

16:3 and/or 18:0 18:1 18:2 18:3

18:4 and/or 20:2 20:0 20:1 20:3 20:4

20:5 and/or 22:2 22:3 22:4 22:5 22:6

Column 1

14-15°C 26-27°C

1.3 1.6 + + + +

14.7 16.0 20.0 19.8

+ + + + 5.4 6.5

31.8 30.8 7.3 7.9 + + O.4 1.0 + + 5.0 5.1 ÷ + 4.0 4.5 1.2 1.2 0,5 0.6 0.4 + 2.1 1.4 5.9 3.6

Column 2

14-15°C 26-27°C

3.9 3.7 1.5 3.1 + +

19.2 22.5 10.1 14.1

+ + +

10.4 7.7 26.6 25.7 15.0 8.0 0.1 1.7 0.8 1.3 0.1 + 2.5 3.6 0.6 0.6 1.5 2.7 0.5 0.7 0.3 + 0.3 + 1.5 0.6 5.1 4.0

Column 3

14-15°C 26-27°C

6.1 4.4 1.7 l . l + +

23.7 24.0 19.0 19.0

+ + + +

12.1 8.6 21.1 24.6

2.4 8.0 1.0 1.1 0.6 0.9 - - + 1.2 1.2 1.1 0.6 2.0 1.4 + 0.5 0.5 0.3 1.0 0.9 + 0.4 6.5 3.0

? Data obtained by gas chromatography of methyl esters. From the work of Knipprath and Mead: °

were reversed. The relative conversions of the two acids lends support to the observation of Reiser and coworkers 74 that fish do not tend to convert 18- carbon acids to long-chain polyunsaturated acids if the precursors are present in the diet at a 5 % level or higher. Significant conversion takes place, however, at a level of 1%.

Further studies with guppies suggested the possibility that both short-term and long-term adaptive mechanisms are active in fish subjected to changes in environmental temperature (Table 8). When fish were forced to adapt to cold water in one day, docosahexaenoic acid reached a maximum in one week and then declined steadily to a near normal level over a period of several weeks. The evidence also suggested that 14:0, 16:1, and 18:2 acids reach higher levels several weeks after forced adaptation. It appears, therefore, that the fatty acid changes occurring immediately after a reduction in environmental temperature may represent a short-term emergency adaptation, primarily via the docosahexaenoic acids, whereas changes taking place in the 14:0, 16:1, and 18:2 acids reflect a long-term adaptive mechanism. Both short- and long-term changes

T H E L I P I D B I O C H E M I S T R Y O F M A R I N E O R G A N I S M S 359

TABLE 8.

Fatty Acid Compositiont of Guppies 2~aised on Complete Trout Chow Diet in Warm Water and Adapted to Cold Water

Fatty acid

14:0 14:1 15:0 16:0 16:1 16:2 16:4

16:3 and/or 18:0 18:1 18:2 18:3

18:4 and/or 20:2 20:2 20:1 20:3 20:4

20:5 and/or 22:2 22:3 22:4 22:5 22:6

2~27°C

8 weeks

3.7 3.1 ÷

22.5 14.1 +

7.7 25.7 8.0 1.7 1.3 ÷ 3.6 0.6 2.7 0.7 + ÷ 0.6 4.0

14--15°C

2 days 8 days 2 weeks 4 weeks

3.0 3.7 3.9 4.6 4.1 3.0 2.1 1.3 + ÷ ÷ ÷

19.8 19.8 19.9 20.0 16.3 14.8 16.8 18.6 + 0.5 + + ÷ ÷ ÷ ÷ 8.5 7.6 6.1 8.5

28.6 26"9 25.4 24.5 8.4 8.7 9.1 9.5 0.7 0.8 1.7 1.6 0.5 0.4 + ÷ - - ÷ ÷ - -

1.4 2.0 2.8 2.8 0.3 0.4 0.9 0.4 1.7 2.2 2.1 1.9 0.7 0.4 1.0 0.7 0.4 0.4 0.6 0.3 0.6 0.6 0.8 0.5 0.6 0.6 0.8 0.3 4.4 7.2 6.0 4.5

$ Data obtained by gas chromatography of methyl esters. From the work of Knipprath and Mead. 4°

might represent an attempt by the organism to maintain proper protoplasmic viscosity, as suggested by Lewis 46 and Mead. 60

Changes in the composition of tissue fatty acids brought about by temperature variations might be explained in terms of a selective absorption of certain structures in the intestine. Reiser and coworkers 74 have suggested that saturated fatty acids, such as lauric and myristic acid, are not absorbed at lower temperatures (13°C) in certain teleost fish. This observation suggests the possibility that environmental temperatures of cold-blooded species might play an important role in the selective absorption of more unsaturated fatty acids at colder temperatures.

2. Pressure (depth) At present, there is not sufficient evidence to allow a precise statement about

the relationship of pressure (depth)to the fatty acid patterns of marine organisms. Studies by Lewis, 4e however, with deepwater fish and crustacea have revealed that certain species are unique for high levels of oleic acid. For example, oleic acid varied from about 15 % in the surface species to 72% in the deepwater


species. Furthermore, medium-chain and polyunsaturated acids appeared to decrease with increasing depth. Unfortunately, the classes of lipids involved were not investigated. The high levels of oleic acid in the deepwater animals might be due to wax esters, such as those reported in several marine species by Nevenzel and coworkers. 64, 65

Future work on the influence of depth on lipid metabolism should involve a consideration of the effects of hydrostatic pressure, per se. Results so far have been clouded by the influence of factors, such as temperature, that are highly variable at different depths in the ocean. Furthermore, with the advent of sophisticated analytical techniques, such as thin-layer chromatography and "stereospecific analysis", 9 interest might profitably be focused in the future on the influence of environmental factors on specific lipid classes. Studies on the specific molecular arrangement of lipids would logically follow from such investigations. We would like to draw attention to the possibility that alterations in the positional distribution of fatty acids in glycerolipids might well take place under the influence of environmental factors, such as temperature and pressure, it seems logical to assume that protoplasmic viscosity, for example, might be regulated via alterations in the positional distribution of fatty acids in glycerolipids, or by the preferential synthesis of certain lipid classes.

C. Stress, sex, and season

Several studies have been reported that relate to the effects of starvation on the lipids offish. The investigations of Wilkins 9~' demonstrated that the mobilization of phospholipids takes place in the herring (Clupea harengus) after pro- longed starvation. Such a finding is supported by the results of Olley 6s with starved cod. The loss of phospholipid from organs during starvation suggests that, under conditions of physiological emergency, phospholipids can be utilized as an energy source. Because of the fundamental role of phospholipids in cellular architecture, the depletion of these compounds under conditions of stress may contribute s.ubstantially to the ultimate death of the organism. Nevertheless, the extensive degeneration of the vascular system during the spawning migrations of trout (Salmo gairdneri) has been shown to regress after spawning. 87 Vascular degeneration, which presumably involves severe alterations in the phospholipids, is not known to regress in other animals. The possibility exists that phospholipids, mobilized and degraded during physiological stress, can be reconstituted as a fundamental part of cellular architecture. Such an idea is not as unreasonable as it might appear because of the unique physiology often related to the adaptive mechanisms of marine organisms.

Several papers have documented changes in fatty acid composition in relation to both sex and season. DeWitt 17 observed that the hepatic lipids of the cod (Gadus callarias) show an increase in polyunsaturated and monoenoic acids during the winter and summer months. An abrupt decline in these acids took place duting the March spawning season, and no significant variation in saturated fatty acids was evident through the annual cycle. Jangaard and


coworkers a5 investigated seasonal changes that occurred in the fatty acids of cod (Gadus morhua) flesh and liver. The fatty acids from the flesh showed no significant changes with respect to either sex or season. However, the hepatic iipids of female fish showed increasing amounts of 20:1 and 22:1 acids as the fat content of the liver increased. Furthermore, the highest levels of these acids were reached in late summer and fall. Other acids did not vary significantly. The male fish, on the other hand, did not exhibit similar seasonal variations.

Interpretation of the results of studies relating sex and season to fatty acid composition are unfortunately complicated by individual differences, such as the sizes of the specimens examined. It would appear that further work is necessary before the influence of sex and seasonal changes on fatty acid composition can be properly evaluated. Future studies require a statistical approach in which such factors as the number of fish, the size, and the sex are appro- priately correlated.

IV. PROSPECTUS

In conclusion, we would like to suggest several areas for future study, even at the expense of being somewhat repetitious. Clearly, advances in marine biochemistry will be limited by our ability to culture marine organisms under closely controlled conditions. In this regard we are delighted to hear of a sym- posium "Use of Fish as Experimental Animals for Basic Research" held at the Department of Biochemistry, University of South Dakota on November 15, 1968.

As our capability to maintain fish stocks for experimental purposes progresses, several important areas of future work might be suggested. For example, the techniques are now available to allow us to explore the relationship of class and structure of lipids to the physiological and biochemical changes brought about in marine organisms by environmental conditions. Studies of this type will take us beyond mere infatuation with matters such as total fatty acid composition. In certain cold-blooded creatures, for example, the content of phospholipid is reported to increase at lower temperatures. 7sa The question might well be asked "Is the incrcase in polyunsaturated acidsat low temperatures primarily brought about by an increase in phospholipids rich in these acids?" The answer to this question, and others related to lipid class and structure, are most important for a deeper understanding of environment-induced effects. Further- more, the influence of the food chain and symbiosis on biochemical alterations is to be encouraged, and of particular interest in these studies would be the elucidation of the relative contribution of the diet and the creature's own metabolism.

It is to be expected that marine life will provide a good selection of new and unusual lipids of interest to the biochemist. Studies of creatures that have not heretofore been examined hold the most hope for fulfilling these ends. It is our belief that marine life holds most promising rewards to those who arc interested in the relationship of lipid composition and biochemistry to the evolution of species.

362 PROGRESS 1N THE CHEMISTRY OF FATS AND OTHER LIPIDS

R E F E R E N C E S

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The lipid biochemistry of marine organisms