Comp. Biochem. Physiol., 1976~ Vol. 5311, pp. 509 to 515. Pergamon Press. Printed in Great Britain
METABOLIC AND HEMATOLOGICAL EFFECTS OF STARVATION IN THE EUROPEAN EEL, A N G U I L L A A N G U I L L A L.--III FATTY ACID COMPOSITION C.~RAN DAVE, MAJ-LIS JOHANSSON-SJOBECK,AKE LARSSON, KERSTIN LEWANDERAND ULF LIDMAN Department of Zoophysiology, University of GiSteborg, Fack, S-400 33 G/Steborg 33, Sweden (Received 19 February 1975) Abstract--1. Fatty acid composition in liver and muscle was studied in the European eel (Anguilla anguilla L.) after 1I, 47, 96 and 164 days of starvation during the winter season. 2. The changes in muscle were small compared with those in liver. 3. In both liver and muscle there was a marked decrease in 14:0.* 4. In the liver starvation caused a decrease in 16:1, 18:1, 20:1 and/or 18:3 and an increase in 20:4, 20:5, 22:4 and 22:5. 5. The observed changes are discussed in relation to differences in distributional pattern among the lipid classes, to ~- and fl-positional preferences and to environmental temperature.
position, but some characteristic differences in their lipid pattern exist. Thus, marine fishes compared to SEVERALreports on fatty acid composition in different freshwater fishes usually have a higher ratio of the species of fish suggest that most fishes have an intrin- linoleic:linolenic type of fatty acids and a higher prosic ability to form a characteristic and complex fatty portion of C16-fatty acids. The reason for this is not acid pattern with large amounts of highly unsaturated fully understood but it might be an effect of different fatty acids (Tashima & Cahill, 1965; Ackman, 1967; diets (Ackman, 1967). Another explanation, suggested Love, 1970). Within this basic pattern the fatty acid by Farkas & Herodec (1964) might be a combined composition may vary with such external factors as effect of feeding habits and annual temperature cycle. diet, temperature and salinity (Lovern, 1938, 1950; The fatty acid patterns of the lipid classes often Love, 1970). vary between different parts or organs of the fish body Diet is certainly the most important factor causing (Addison et al., 1968; Kluytmans & Zandee, 1973b; alterations in the fatty acid pattern of the fish lipids. Dave et al., 1974). Dietary influence on fatty acid composition has been Studies on the fatty acid distribution between the shown in eels, Anguilla spp. (Lovern, 1938, 1940; Arai ~- and fl-positions of the triglycerides and lecithins et al., 1971), as well as in other fish species (Reiser in liver and muscle of the cod, Gadus callarias, have et al., 1963; Kaneko et al., 1966). further shown that the polyunsaturated fatty acids of The influence of environmental temperature on the the linolenic type were preferably located in the /~fatty acid composition and especially on the degree position (Brockerhoff et al., 1963). The same kind of of unsaturation in the body lipids has been demon- positional preference has also been shown in total strated in fishes as well as in other poikilotherms oil triglycerides of the North American eel, Anguilla (reviewed by Hazel & Prosser, t974). Generally rostrata (Brockerhoff & Hoyle, 1963). It was suggested speaking, a lowered temperature produces an in- that this preference would conserve the polyunsaturcreased proportion of unsaturated fatty acids. Effects ated fatty acids of marine organisms by virtue of the of temperature on the lipid composition have been retention of the fl-monoglyceride structure. shown in the eel, Ar~uilla an(tuilla L. (Lovern, 1950) Only a few earlier studies have dealt with the effects and most extensively in the goldfish, Carassius auratus of starvation on the fatty acid composition in fish. (Hoar & Cottle, 1952; Knipprath & Mead, 1967; Kaneko et al. (1966) found that the proportion of Johnston & Roots, 1964; Roots, 1968; Roots & Johnhighly unsaturated fatty acids in the muscle lipids of ston, 1968; Kemp & Smith, 1970; CaldweU & Vern- rainbow trout, Salmo irideus, increased during starvaberg, 1970). Combined effects of diet and temperature tion for 10 weeks. The same trend towards an increase have also been studied (Lovern, 1950; Reiser et al., in the highly unsaturated fatty acids was observed 1963; Farkas & Herodec, 1964). in the Northern pike, Esox lucius L., starved for 2 Marine and freshwater fishes certainly have the months (Kluytmans & Zandee, 1973a). The latter same mechanisms for regulating their fatty acid com- authors, however, concluded that the differences in fatty acid composition of the total lipids (excluding * Number of carbon:number of double bonds in the liver and gonads) were small between regularly fed fatty acid, fishes and starved fishes. 509 INTRODUCTION
G(~IRAN DAVIi et al.
510
Many fish species have a natural starvation period during the winter season. The purpose of this study was to describe the effects of such starvation on the fatty acid composition of the European eel, Al~guilla anguilla L. Results on metabolic and hematological alterations during this starvation study are presented in two parallel studies (Dave et al., 1975; JohanssonSi~beck et al., 1975). MATERIALS AND METHODS
Detailed information about animal capture, experimental conditions and sampling procedures, as well as the method for lipid extraction (according to Carlsson, 1963) are presented in a parallel study (Dave et al., 1975). The present ratios between the lipid classes in liver and muscle tissue were calculated from the individual amounts (expressed as mg/l(X)mg wet wt) of triglycerides, phospholipids and cholesterol obtained in the parallel study.
Methylatio,1 o / l i p i d s 2 ml of lipid extract was transferred to a glass stoppered test tube and evaporated to dryness under reduced pressure. Then 2m] of 0-5 M sulfuric acid (Merck P.A.) in methanol (Merck P.A.) was added to the tube. After 3 days at room temperature I ml of n-heptanc (Mallinckrodt A.R.) was added and the solution was thoroughly mixed and allowed to stand for 1 hr. 2 ml of distilled water was added and the tube was stored (1 10 days) in the refrigerator ( + 4 C ) until analysed by gas liquid chromatography (GLC) for fatty acid composition. Gas liquid chromatography (GLC) The GLC-analysis was performed on a Perkin-Elmer F I1 instrument of an all-glass, dual column type, equipped with two flame-ionization detectors and a Perkin Elmer 165 recorder. The two columns were prepared by packing glass tubes (5ft x-~in.) with HI-EFF IBP (diethylene glycol succinate), 157;i on 80/100 mesh Chromosorb W (AW) pretested packing (Applied Science Laboratories Inc., State College, Pa., U.S.A.). The following operation conditions were used: Temperaturesof column and injection block were 180 and 260C respectively. Carrier gas: Nitrogen (extra pure) at a flow rate of 30 ml/min. For the analysis 1 #1 of the heptane phase (upper phase), containing the methyl esters of the fatty acids, was injected. The identification of the fatty acids was made by comparing retention times of actual standards and by calculating, according to Ackman (1965), the relative retention times to oleic acid (18:1). For quantification the height (H) of each determined peak was measured to the nearest 1 mm, and the width at the half height (W~ 2H) was measured to the nearest 0.1 mm by the aid of a pair of vernier callipers. The area of each peak (H x W , ; n) and the percentage distribution were calculated without any correction factors. Minor components were excluded in the quantification. Hydrogenation of methyl esters After analysing the fatty acid composition of the methylated lipid extract from liver tissue some Platinum-IV oxide (Gelhard, Stockholm, Sweden) was added to the heptane phase containing the methyl esters. The tubes were then filled with hydrogen gas at a pressure of 2 atm and allowed to stand overnight. 1/*1 of the upper phase was injected for GLC. Identification and quantification were performed as above. Statistical treatment The data after 47, 96 and 164 days were tested against those after l 1 days and also against those from the preceding sampling occasion. All statistical tests were made by
Student's t-test. Significant differences were established at the 0-05-level. RESULTS AND DISCUSSION
The effects of starvation on muscle and liver triglyceride-phospholipid (TG-PL) ratio, phospholipid cholesterol ( P L - C H O L ) ratio, percentage of myristic acid (14:0) and on the percentage of liver total C~4fatty acids (after hydrogenation) are presented in Fig. 1. The effects on C16-, C,s-, C2o- and C2_~-fatty acids are presented in Figs. 2, 3, 4 and 5 respectively.
Triglycerides, phospholipids a~d cholesterol The absolute percental changes (w/w) in triglycerides, phospholipids and cholesterol are presented elsewhere (Dave et al., 1975). As the analysis of the fatty acid composition in this study was made on total lipid extracts, the relative proportions are more important than the absolute amounts of the different lipid classes. According to Addison et al. (1968) the fatty acid composition of the different lipid classes showed great differences in muscle from the cod, Gadus morhua. Triglycerides compared to the other lipid classes contained more 14:0, 16:1, 18:1, 20:1 and 22:1 and less 20:5 and 22:6. Muscle triglycerides also closely resembled the total lipids in liver as regards fatty acid composition. In a study on the Northern pike, Esox lucius L., Kluytmans & Zandee (1973b) obtained somewhat different results, but also these authors found that 18:1 was more abundant in the triglyceride fraction than in other lipid fractions including monoand diglycerides. The latter is in line with the observation by Brockerhoff & Hoyle (1963) that in fish oil 12 I
T G - P L r01"io (
4~ (H)
(~)
2/
i
r
(8)
P L - CHOL.rotio
h
Muscle Z*I4:0 (u) I
{8) I
Liver
--{ PL-CHOL rotio I
02)
(8)
~,~
~,2~z~----~ ....
"*TG-
PL
rQl"io
Liver I* (lO) ]
II
47
001 f 96
114:0 i
Liver 4 " 14 : tol-a I 164
Doys
Fig. 1. Effects of starvation on fatty acid composition in the eel. Triglyceride-phospholipid (TG PL) ratio, phospholipid-cholesterol (PL-CHOL) ratio in liver and muscle tissue. Myristic acid (14:0) in muscle and liver total lipids (%). Total C14-fatty acids (14:total) in liver lipids ('},% Results are expressed as mean +_ S.E. Number of fishes in parentheses; *P < 0.05 when tested against the results after 11 days; **P < 0'05 when tested against the results from the preceding sampling event.
Effects of starvation in the European eel triglycerides 18:1 is more abundant in the a-position than in the fl-position. In the latter study it was also e I ~l) found that fish triglycerides, like lecithins of most vertebrates, preferably store the polyunsaturated fatty acids in the//-position. The assumption has also been 20 ( ) (111) ~)16:O made that the polyunsaturated acids can be retained in the //-position in the lipid metabolism along the 12 I ~ Liver food chains by means of the chemical stability of the Io ( 16:1 fl-monoglyceride structure (Brockerhoff et al., 1963). In the present study the triglyceride-phospholipid (TG-PL) ratio was initially (11 days) about six times .Liver higher in muscle compared to liver (Fig. 1). As shown 26 ~ 16;O in a parallel study (Dave et al., 1975) the triglyceride content decreased more in muscle compared to liver, 24 ) but the decrease in T G - P L ratio in muscle between l 22 ~ (~o) 11 and 96 days was only 33% while the decrease in I (12) liver was more Ihan 50%. This more pronounced (12) 20 reduction in TG PL ratio in the liver during starvaI I tion was also accompanied by greater changes in fatty Liver acid composition in this tissue compared t6 muscle 30 16 : total (8) tissue. 1 28 The phospholipid-cholesterol (PL-CHOL) ratio (12) 1 I was of the same magnitude in liver and muscle. The 96 164 47 fluctuations in this ratio were almost parallel in liver Doys Fig. 2. Effects of starvation on fatty acid composition in and muscle during starvation (Fig. 1), For muscle the the eel. Palmitoleic acid (16:1) and palmitic acid (16:0) increase between 47 and 96 days was significant in muscle and liver total lipids (%). Total C16°fatty acids (P < 0-05). This effect might be due to changes in (16:total) in liver total lipids (%). environmental temperature. Thus the increased PLResults are expressed as mean + S,E. Number of fishes CHOL ratio between 47 and 96 days can he explained in parentheses; *P < 0.05 when tested against the results by the reduced muscle cholesterol content, which after 11 days; **P < 005 when tested against the results occurred contemporarily with a decline in water temfrom the preceding sampling event. perature (Dave et al., 1975). The change in muscle cholesterol content might be a result of an altered ~(12) (lit 3 ) Muscle
,of 02)
~ cult
Musc,e 16:1
I
361 I
t
51-
*~**
I
I Muscle
6~ (12)
(lit
(11)
(8) Muscle
[ (~)
?
~,~t
(8,)Musc,e
36
Liver
I
34
18:1
i| I 4~
I (tl)
I 20:4 Is) Muscle
3]-(ij2)~'''~ (~l)
(ll~l)
J
(8)]18:0
02)
(~) .....---[
~ 20:1
6t"30
4
28
26
)
I
6
I
~
I
(
4 [- ~
40 { 32 ~
) {'P)
18:0Liver
1121~
Liver
18 : total
56
32
I
I
96 Days
l
j-* Liver 20 : 5
)
I
*
4
61-~.'~
!,,2t
.Liver 20:4
~
)
2f ( 0 141]2f II
47
1-
o
~
,
[8)
,
~
,
Liver .20:1
*///._._~.~* //~c~
~Oi-Ozt/I / ~P~ t,2)
) I
~
~I-
' ~ [ - -
34
(
/ I
I 6
(8)
~
(
47
I
96
Liver (,) 20 : total I
164
Days
164
Fig. 3. Effects of starvation on fatty acid composition in the eel. Oleic acid (18:1) and stearic acid (18:0) in muscle and liver total lipids (%). Total Cla-fatty acids (18:total) in liver total lipids (%). Results are expressed as mean + S.E. Number of fishes in parentheses; *P < 0.05 when tested against the results after 11 days; **P < 0"05 when tested against the results from the preceding sampling event.
Fig. 4. Effects of starvation on fatty acid composition in the eeL Eicosapentaenoic acid (20:5), eicosatetraenoic acid (20:4) and ¢icosenoic 120:1) and/or linolenic acid 08:3) in muscle and liver total lipids (%). Total C20-fatty acids (20:total) in liver total ]ipids (%). Results expressed as mean _+ S.E. Number of fishes in parentheses; *P < 0.05 when tested against the results after 11 days; **P < 0-05 when tested against the results from the preceding sampling event.
512
GORAN DAVE et al.
I 5 L| (~_ 3 '
1,0 I (u) ] f
~
Mu,c,e
I
I~l 22 : 5 I
m) I I
(s) Muscle ! 22:4
161 ~ 1 4
~(8)
Liver 22:5
12 (i2) i0
F'~
!
(iIo)
I
~
24
Liver 22:4
(
) I
I
I Liver 22: totol
~ *
22
20
/
(~)
16 ( 14 1 III
Oz) 47I
1 96 Days
I 1£;4
Fig. 5. Effects of starvation on fatty acid composition in the eel. Docosapentaenoid acid (22:5} and docosatetraenoic acid (22:4) in muscle and liver total lipids (.,,). Total Ce2-fatty acids (22:totalt in liver total lipids (%{i)Results are expressed as mean Jr S.E. Number of fishes in parentheses; *P < 0.05 when tested against the results after 11 days; **P < 0.05 when tested against the results • from the preceding sampling event. activity in plasma lecithin-cholesterol acyltransferace (LCAT). This role of LCAT, i.e. performing changes in cholesterol content of biological membranes and thus changing their properties, is suggested to be its main physiological role in mammals (Glomset, 1970). The present results suggest that a similar role of LCAT can be valid also in fish. 14:0 and 14: total.latty acids
The decrease in 14:0 in both liver and muscle (Fig. 1) was almost parallel with the decrease in T G PL ratio (except for muscle after 164 days). This is in line with the finding of Addison et al. (1968) that 14:0 is found mainly in the triglyceride fraction• The values for 14: total in liver tissue showed a successive decrease during the starvation period. When comparing the present results on liver 14:0 and liver 14: total (after hydrogenation) it is observed that the values of 14:0 exceeded those of 14: total. This is theoretically impossible and must be due to a more accentuated overestimation of earlier peaks compared to later peaks in the GLC analytical procedure for the non-hydrogenated than for the hydrogenated samples. In the former the polyunsaturated fatty acids are represented by later and broader peaks. Furthermore, myristoleic acid (14:1), which only constituted a minor component in the eel fat, was not quantified at any occasion as no detectable changes in this fatty acid were observed. Accordingly, 14:1 is not likely to contribute to any observed change in 14: total and therefore the latter parameter can be regarded as a
more accurate estimation of the content of 14:0 in liver tissue. 16:0. 16:1 and 16 : total fittly acids
In muscle lipids from the cod 16:1 had a clear preference for the triglyceride fraction, while 16:0 had a more complex distribution in different lipid classes (Brockerhoff et al., 1963). In fish triglycerides neither 16:0 nor 16:1 fatty acids did not appear to show any particular preference for :~- or fl-position (Brockerhoff & Hoyle, 1963), while in liver and muscle phosphatidyl choline of the cod both 16:(t and 16:l showcd a marked preference for the >position (Brockerhoff et al.. 1963). In the present study the starvation did not causc any changes either in muscle 16:0 and 16:1. or in liver 16: total thtty acids. On the other hand, there was a continuous decrease in liver 16:1 and a parallel increase in 16:0 (Fig. 2). This combined effect of starvation on liver 16: fatty acids is hard to explain on the basis of different distribution in lipid classes or of preferential ~- or fi-positions. Thc effect is therefore more likely to be seen as a reduced olefin synthesis ability in the starving animal. Such an assumption is supported by results obtained in studies on fasting rats (Elovson, 1965; Benjamin & Gellhorn, 1966: kec & Sprecher. 1971). 1~:0, 18:1 and 18: total/arty acids
According to Addison et al. (1968) 18:0 showed a relatively even distribution among most of the different lipid classes in cod muscle. One exception was serine phosphoglyceride, which had a content o1" 18:0 3 6 times higher than the other lipid classes. 18:t. on the other hand, showed some preference for the triglyceride fraction. The latter finding has also been observed in the Northern pike (Kluytmans & Zandee, 1973b). These authors observed that the triglyeeride fraction was relatively poor in IS:0. Initially (11 daysl the amounts of both 18:0 and 18:1, expressed as percentage of total estimated fatty acids, were equal in liver and muscle tissue. However. the individual variations, indicated by the standard errors (S.E.), were more pronotinced in liver compared to muscle tissue lFig. 3). The levels of 18:0 and 1~:1 in lnuscle tissue were relatively unaffected during starvation except for the slight increase in 18:0 between 11 and 47 days. The liver, on the other hand, showed a pronounced decrease in 18:1 and 18: total, while the level of 18:0 was almost unchanged. These findings, together with other observations, suggest: 1. A pronounced difference in lipid metabolism in muscle and liver. 2. A decreased olefin synthesis in the liver ot the starving eel. 3. Compared to other fatty acids, 18:1 appears to provide the most energy for metabolic purposes in lasting eels. The first suggestion is based on the fact that the levels of 1g:0 as well as 18:1 were equal in liver and muscle initially (11 days), but then the utilization of preferential fatty acids, like 18:1, is much more pronounced in the liver although the lipid class utilized mainly in both organs is triglycerides (Fig. l j. The
Effects of starvation in the European eel second suggestion agrees with similar findings for 16:1 in the present study and is further supported by results obtained in fasting mammals (Elofson, 1965; Benjamin & Gellhorn, 1966; Lee & Sprecher, 1971; Seedy & Poole, 1974). The third suggestion is based on the findings that the decrease in liver 18:1 was not accompanied by any comparable increase in 18:0. Instead the liver 18: total content diminished. Furthermore, the increase in liver 16:0 cannot explain the decrease in both 16:1 and 18:1. Thus, the reduced liver 18:1 cannot solely be due to a decreased olefin synthesis. The assumption that the primarily utilized fatty acid was 18:1 is also in agreement with the observation that 18:1 seemed to be the most preferably located fatty acid in the ~t-position in the liver triglycerides of the American eel, Anouilla rostrata (Brockerhoff & Hoyle, 1963). Therefore 18:1 is, to a higher degree than the other fatty acids, likely to be hydrolysed from the triglyceride molecule and enter the d-oxidation for energy purposes. Probably this is valid also for other vertebrates. Thus, it has been reported that 18:1 was the fatty acid showing the greatest decrease in carcass fat during starvation in the neonatal swine (Seedy & Poole, 1974). 20:1 and/or 18:3, 20:4, 20:5 and 20: total fatty acids In the muscle lipids of cod, Addison et al. (1968) found that 20:1 was about three times more abundant in triglycerides than in other lipid classes. In addition 18:3 and 20:4 showed no obvious preference for any special lipid class, while 20:5 was two to three times more abundant in sterol esters and choline phosphatides than in other lipid classes. Kluytmans & Zandee (1973b) observed preference of all these fatty acids in triglycerides compared to phospholipids in the Northern pike. In the triglycerides of the American eel, Anouilla rostrata, 20:1 showed preference for the a-position, while 20:5 together with 20:3 and/or 20:4 showed preference for the d-position (Brockerhoff & Hoyle, 1963). A similar preferential distribution was also observed in phosphatidyl choline of cod liver and muscle, as well as in muscle of the scallop, Plactopecten ma#ellanicus, and liver of the lobster, Homarus americanus (Brockerhoff et al., 1963). In the present study no significant changes were observed in muscle C2o fatty acids (Fig. 4) in spite of changes in the T G - P L ratio. In the liver, on the other hand, there was a marked increase in 20:4 and 20:5 and a marked decrease in 20:1. 20: total fatty acids increased in the liver during starvation. The changes in C2o fatty acids of the liver can certainly be explained by various distributional patterns of these fatty acids in the different lipid classes and ~or d-positional preference as described above. The disagreement between changes in C2o-fatty acids in" liver and muscle might therefore be due to different regulation of the lipid metabolism in liver and muscle. In the eel the main part of the depot fat is stored in the muscle (Dave et al., 1975), which showed very limited alterations in the fatty acid pattern. Accordingly there is no profound change in fatty acid pattern of the whole fish. Therefore deficiency symptoms caused by lack of polyunsaturated fatty acids are not likely to occur during starvation in the eel. Deficiency symptoms like impairment of growth and
513
pigmentation have previously been reported in fry of chinook salmon, Oncorhynchus tshawytscha, fed a fatfree diet for 24 weeks (Nicolaides & Woodall, 1962). 22:4, 22:5 and 22: total fatty acids The identification of polyunsaturated fatty acids, generally occurring in fish lipids, by means of the retention time after GLC analysis, is mostly unprecise, especially regarding the degree of unsaturation. Comparisons between different studies should therefore be made with great caution. In the present study no efforts were made to identify the fatty acids by other means than GLC before and after complete hydrogenation. Accordingly only the number of carbon atoms is determined more precisely while the number of double bonds is more uncertain. The starvation did not cause any significant alteration in the content of 22:4 and 22:5 in muscle tissue (Fig. 5). On the other hand, in liver tissue 22:4 increased during the first 96 days, while 22:5 and 22: total showed a successive elevation during the progress of the starvation. With reservation for the difficulties mentioned above, it is yet likely that the fatty acids identified in the present study as 22:4 and 22:5 are the same as those identified by other authors (Addison et al., 1968) as 22:5 and 22:6 respectively. These two acids have been found to be mainly located in non-triglycerides of cod muscle lipids (Addison et al., 1968). In the triglycerides of the American eel, Anguilla rostrata, they were further found to be preferably located in the d-position (Brockerhoff & Hoyle, 1963). Such reasons are probably responsible for the percental increase in 22:4 and 22:5 in the liver, as the phospholipids are regarded as structural elements in contrast to triglycerides, and the fatty acids in the d-position are not as easily hydrolysed as those in the or-position.
CONCLUSIONS
Starvation in the European eel, Anguilla anguilla, has a pronounced influence on the fatty acid composition of the liver. The muscle, which is the main fat depot in the eel, on the other hand, shows much smaller alterations in fatty acid composition. The changes in fatty acid composition can be partly explained by altered ratios between the different lipid classes, which in other aquatic animals have been shown to have differences in fatty acid pattern (Addison et al., 1968; Kluytmans & Zandee, 1973b). Also preferential location in ~- or fl-position in triglycerides and lecithins (Brockerhoff et al., 1963; Brockerhoff& Hoyle, 1963) might contribute to some of the observed changes. These reasons might be valid for the continuous decrease in 14:0 in both liver and muscle and the increase in liver polyunsaturated acids (20:4, 20:5, 22:4 and 22:5). Disagreement of the starvation-induced changes in the fatty acid pattern between liver and muscle suggests a different regulation of the lipid metabolism in these two tissues. The changes in the monoenes (16:1, 18:1 and 20:1 and/or 18:3) in liver tissue are in line with findings on mammals that olefin synthesis is almost completely abolished during starvation. Studies on rats (Elovson, 1965) have further shown
514
GORAN DAWz et al.
that in extrahepatic tissues desaturation a n d elongation of fatty acids are less p r o m i n e n t than in liver. The more p r o n o u n c e d changes in liver c o m p a r e d to muscle might be a n indication that also in the eel the liver is the main organ for fatty acid metabolism (synthesis, interconversion a n d oxidation). The transient increase in muscle p h o s p h o l i p i d cholesterol ratio observed at 96 days (Fig. 1) might be a response to the lower water temperature by this time. Thus, the lowered muscle cholesterol content after 96 days would, as cholesterol is considered as mainly a m e m b r a n e constituent, adjust the fluidity of the membranes. This m e c h a n i s m of adjusting the m e m b r a n e properties in relation to temperature seems more probable than alterations in the proportions of saturated a n d polyunsaturated fatty acids. The changes in fatty acid pattern of the liver lipids are very similar to observations made during cold acclimation, i.e. an increase in polyunsaturated fatty acids. However, the only fatty acid showing some relation to water temperature (presented by Dave et al., 1975) was 22:4, while the other polyunsaturated fatty acids (20:4, 20:5 a n d 22:5) did not. In studies on temperature influence on the fatty acid composition in fish the nutritional state must therefore be considered carefully, as m a n y fish species, including the eel, lose appetite when the temperature is lowered. The percental a m o u n t s of myristic acid (14:0), palmitoleic acid (16:1) a n d oleic acid (18:1) in liver lipids decline almost continuously during starvation and might serve as an indication of the nutritional state in the eel a n d possibly also in other tish species. Acknowled,qements The authors wish to thank Mrs. Sir ()stling for her excellent technical assistance during this work. We also wish to thank Dr. Bertil Swedmark at Kristineberg Zoological Station. The financial support of the National Board of Fisheries, Sweden, is also gratefully acknowledged. REFERENCES
ACKMAN R. G. (1965) Cod liver oil as secondary standards in the GLC of polyunsaturated fatty acids of animal origin: analysis of a dermal oil of the Atlantic leatherback turtle. J. Am. Oil Chem. Soc. 42, 38 42. ACKMAN R. G. (1967) Characteristics of the fatty acid composition and biochemistry of some fresh-water fish oils and lipids in comparison with marine oils and lipids. Comp. Biochem. Physiol. 22, 907 922. ADDISON R. F., ACKMAN R. G. & HINGLEY J. (1968) Distribution of fatty acids in cod flesh lipids. J. Fish Res. Bd Can. 25, 2083--2090. ARM S., NOSE T. & HASHImOtO Y. (1971) A purified test diet for the eel, Anguilla japonica. Bull. Freshwater Fish. Res. Lab. 21, 161 178. BENJAMIN W. & GELLHORN A. (1966) RNA biosynthesis in adipose tissue: effect of fasting. J. Lipid Res. 7, 285294. BROCKERHOFF H., ACKMAN R. G. & HOYLE R. J. (1963) Specific distribution of fatty acids in marine lipids. Arch. Bioehem. Biophys. 100, 9 12. BrOCKERHOFF H. & HOYLE R. J. I1963) On the structure of the depot fats of marine fish and mammals. Arch Biochem. Biophys. 102, 452-455. CALDWELL R. S. & VERNBER6 J. F. (1970) The influence of acclimation temperature on the lipid composition of fish gill mitochondria. Comp. Bioehem. Physiol. 34. 17% 191.
CARLSSON L. A. (1963) Determination of serum triglycerides. J. Atheroscler. Res. 3, 334~336. DAVE G., JOHANSSON M.-L., LARSSON A., LEWANDER K. & LIDMAN U. (1974) Metabolic and hematological studies on the yellow ane silver phases of the European eel, 4nguilla anyuilla L. II. Fatty acid composition. Comp. Biochem. Physiol. 47B, 583 591. DAVI! G., JOHANSSON-Sf)BECK M.-L., LARSSON /~.. LEWANDI~P,K. & LIDMAN U. {1975) Metabolic and hematological effects of starvation in the European eel, Amduilla amluilla L. h Carbohydrate, lipid, protein and inorganic ion metabolism. Comp. Biochem. Physiol. 52A, 423 430. ELovsox J. (19651 Conversions of palmitic and stearic acid in the intact rat. Biochim. hiophys. Acta 106, 291 303. FAr~ZAST. & HErODEC S. (1964) The effect of environmental temperature on the fatty acid composition of crustacean plankton, J. Lipid Res. 5. 369 373, GLOMS~iTJ. A. (1971)) Physiological role of lecithin cholesterol acyltransferase. Anl..I. clin. Chem. 23, 1129-1136. HAZH, J. R. & ProssEr C. L. (1974} Molecular mechanisms of temperature compensation in poikilotherms. Physiol. Ret..54 {3), 596619. HOAR W. S. & Cor'rLE M. K. (1952) Some effects of temperature acclimatization on the chemical constitution of goldfish tissues. Can. J. Zool. 30, 49 54. JOHANSSON-SJI~BI!CK M.-L., DAVE G., LARSSON ,~., L1WANDIR K. & LIDMAN U. 11975) Metabolic and hematological effects of starvation in the European eel. Anguilla an¢luilla L. II. Hematologs,. Comp. Biochem. Physiol. 52A. 431 434. JoH~,srox P. V. & Roots B. 1. (1964) Brain lipid lhtt2r acids and temperature acclimation. Comp. Biochem. Physiol. II. 303 309. KANI!KO T., TAKI-UCHI M,. ISHll S., HIGASHI H. 8¢~ KIKUCHI T. (1966) Effect of dietary lipids on fish under cultivation IV. Changes of fatty acid composition in flesh lipids of rainbow trout on nun-feeding. Bull. Jap. Soc. Sciem. F'ish. 33, 56 58 (abstract in English). KtMp P. & SrvHt;t M. W. [1970) Effect of temperature acclimatization on the fatty acid composition of goldfish intestinal lipids. Bioehem. J. 117, 9 15. KLt;;'TMANS J. H. F. M. & ZaYDtl: D. 1. (1973a) Lipid metabolism in the Northcrn pike (Esox lucius L.} -I. The fatty acid composition of the Northern pike. Comp. Biochem. Physiol. 44B, 451 458. KLUYTMANS J. H. F. M. & ZANDEE D. J. 11973b) Lipid metabolism in the Northern pike (Esox lucius k.) II. The composition of the total lipids and of the fatty acids isolated from lipid classes and some tissues of the Northern pike. Comp. Biochem. Physiol. 44B, 459 466. KNU'PRATtt W. G. & MEAD J. F. (1967) The effect of environmental temperature on the fatty acid composition and on the i~ vivo incorporation of [1-~4C]-acetate in goldfish (Carassius auratus L.). Lipids 3. 121 128. LEk C.-J. & SP~¢EC'HERH. 11971) An in vitro study of the efl'ects of dietary alterations and fasting on the desaturation of palmitic, stearic eicosa-8,11-dienoic and eicosa8,11,14-trienoic acids. Bioehim. hiophys. Acta 248, 180 185. LOVE R. M. (1970) The Chemical Biolo~ty o/ Fishes, pp. 216 217. Academic Press, New York. Lovers J. A. (1938) Fat metabolism in fishes--Xlll. Factors influencing the composition of the depot fat o[" fishes. Biochem. J. 32, 1214-1224. LOW:RN J. A. (1940) Fat metabolism in fishes. The utilization of the ethyl esters of fatty acids by the eel and their effect on depot fat composition. Biochem. J. 34, 70,-~708. LOVI-:RN J. A. (1950l Some causes o[" wmation in the composition of fish oils. J. Soc. Leath. 7i'ades Chem. 34, 7 21. NICOLAn)ES N. & WoOI)AEl A. N. {1962) lmpaired pig-
Effects of starvation in the European eel mentation in chinook salmon fed diets deficient in essential fatty acids. J. Nutr. 78, 431-437. REISER R., STEVENSONB., KAYAMAM., CHOUDHURYR. B. R. & HOOo D. W. (1963) The influence of dietary fatty acids and environmental temperature on the fatty acid composition of teleost fish. J. Am. Oil Chem. Soc. 40, 507-513. ROOTSB. J. (1968) Phospholipids of goldfish (Carassius auratus L.) brain: the influence of environmental temperature. Comp. Biochem. Physiol. 25, 457-466.
515
ROOTS B. J. & JOHNSTONP. V. (1968) Plasmalogens of the nervous system and environmental temperature. Comp. Biochem. Physiol. 26, 553-560. SEERLEYR. W. • POOLE D. R. (1974) Effect of prolonged fasting on carcass composition and blood fatty acids and glucose of neonatal swine. J. Nutr. 104, 210-217. TASHIMAL. & CAHILLG. F. (1965) Fat metabolism in fish. In Handbook of Physiology (Edited by RENOLD A. E. & CAHILLG. F. JR.), Sect. 5, Adipose tissue, pp. 5~58. American Physiological Society, Washington, D.C.
METABOLIC AND HEMATOLOGICAL EFFECTS OF STARVATION IN THE EUROPEAN EEL, A N G U I L L A A N G U I L L A L.--III FATTY ACID COMPOSITION C.~RAN DAVE, MAJ-LIS JOHANSSON-SJOBECK,AKE LARSSON, KERSTIN LEWANDERAND ULF LIDMAN Department of Zoophysiology, University of GiSteborg, Fack, S-400 33 G/Steborg 33, Sweden (Received 19 February 1975) Abstract--1. Fatty acid composition in liver and muscle was studied in the European eel (Anguilla anguilla L.) after 1I, 47, 96 and 164 days of starvation during the winter season. 2. The changes in muscle were small compared with those in liver. 3. In both liver and muscle there was a marked decrease in 14:0.* 4. In the liver starvation caused a decrease in 16:1, 18:1, 20:1 and/or 18:3 and an increase in 20:4, 20:5, 22:4 and 22:5. 5. The observed changes are discussed in relation to differences in distributional pattern among the lipid classes, to ~- and fl-positional preferences and to environmental temperature.
position, but some characteristic differences in their lipid pattern exist. Thus, marine fishes compared to SEVERALreports on fatty acid composition in different freshwater fishes usually have a higher ratio of the species of fish suggest that most fishes have an intrin- linoleic:linolenic type of fatty acids and a higher prosic ability to form a characteristic and complex fatty portion of C16-fatty acids. The reason for this is not acid pattern with large amounts of highly unsaturated fully understood but it might be an effect of different fatty acids (Tashima & Cahill, 1965; Ackman, 1967; diets (Ackman, 1967). Another explanation, suggested Love, 1970). Within this basic pattern the fatty acid by Farkas & Herodec (1964) might be a combined composition may vary with such external factors as effect of feeding habits and annual temperature cycle. diet, temperature and salinity (Lovern, 1938, 1950; The fatty acid patterns of the lipid classes often Love, 1970). vary between different parts or organs of the fish body Diet is certainly the most important factor causing (Addison et al., 1968; Kluytmans & Zandee, 1973b; alterations in the fatty acid pattern of the fish lipids. Dave et al., 1974). Dietary influence on fatty acid composition has been Studies on the fatty acid distribution between the shown in eels, Anguilla spp. (Lovern, 1938, 1940; Arai ~- and fl-positions of the triglycerides and lecithins et al., 1971), as well as in other fish species (Reiser in liver and muscle of the cod, Gadus callarias, have et al., 1963; Kaneko et al., 1966). further shown that the polyunsaturated fatty acids of The influence of environmental temperature on the the linolenic type were preferably located in the /~fatty acid composition and especially on the degree position (Brockerhoff et al., 1963). The same kind of of unsaturation in the body lipids has been demon- positional preference has also been shown in total strated in fishes as well as in other poikilotherms oil triglycerides of the North American eel, Anguilla (reviewed by Hazel & Prosser, t974). Generally rostrata (Brockerhoff & Hoyle, 1963). It was suggested speaking, a lowered temperature produces an in- that this preference would conserve the polyunsaturcreased proportion of unsaturated fatty acids. Effects ated fatty acids of marine organisms by virtue of the of temperature on the lipid composition have been retention of the fl-monoglyceride structure. shown in the eel, Ar~uilla an(tuilla L. (Lovern, 1950) Only a few earlier studies have dealt with the effects and most extensively in the goldfish, Carassius auratus of starvation on the fatty acid composition in fish. (Hoar & Cottle, 1952; Knipprath & Mead, 1967; Kaneko et al. (1966) found that the proportion of Johnston & Roots, 1964; Roots, 1968; Roots & Johnhighly unsaturated fatty acids in the muscle lipids of ston, 1968; Kemp & Smith, 1970; CaldweU & Vern- rainbow trout, Salmo irideus, increased during starvaberg, 1970). Combined effects of diet and temperature tion for 10 weeks. The same trend towards an increase have also been studied (Lovern, 1950; Reiser et al., in the highly unsaturated fatty acids was observed 1963; Farkas & Herodec, 1964). in the Northern pike, Esox lucius L., starved for 2 Marine and freshwater fishes certainly have the months (Kluytmans & Zandee, 1973a). The latter same mechanisms for regulating their fatty acid com- authors, however, concluded that the differences in fatty acid composition of the total lipids (excluding * Number of carbon:number of double bonds in the liver and gonads) were small between regularly fed fatty acid, fishes and starved fishes. 509 INTRODUCTION
G(~IRAN DAVIi et al.
510
Many fish species have a natural starvation period during the winter season. The purpose of this study was to describe the effects of such starvation on the fatty acid composition of the European eel, Al~guilla anguilla L. Results on metabolic and hematological alterations during this starvation study are presented in two parallel studies (Dave et al., 1975; JohanssonSi~beck et al., 1975). MATERIALS AND METHODS
Detailed information about animal capture, experimental conditions and sampling procedures, as well as the method for lipid extraction (according to Carlsson, 1963) are presented in a parallel study (Dave et al., 1975). The present ratios between the lipid classes in liver and muscle tissue were calculated from the individual amounts (expressed as mg/l(X)mg wet wt) of triglycerides, phospholipids and cholesterol obtained in the parallel study.
Methylatio,1 o / l i p i d s 2 ml of lipid extract was transferred to a glass stoppered test tube and evaporated to dryness under reduced pressure. Then 2m] of 0-5 M sulfuric acid (Merck P.A.) in methanol (Merck P.A.) was added to the tube. After 3 days at room temperature I ml of n-heptanc (Mallinckrodt A.R.) was added and the solution was thoroughly mixed and allowed to stand for 1 hr. 2 ml of distilled water was added and the tube was stored (1 10 days) in the refrigerator ( + 4 C ) until analysed by gas liquid chromatography (GLC) for fatty acid composition. Gas liquid chromatography (GLC) The GLC-analysis was performed on a Perkin-Elmer F I1 instrument of an all-glass, dual column type, equipped with two flame-ionization detectors and a Perkin Elmer 165 recorder. The two columns were prepared by packing glass tubes (5ft x-~in.) with HI-EFF IBP (diethylene glycol succinate), 157;i on 80/100 mesh Chromosorb W (AW) pretested packing (Applied Science Laboratories Inc., State College, Pa., U.S.A.). The following operation conditions were used: Temperaturesof column and injection block were 180 and 260C respectively. Carrier gas: Nitrogen (extra pure) at a flow rate of 30 ml/min. For the analysis 1 #1 of the heptane phase (upper phase), containing the methyl esters of the fatty acids, was injected. The identification of the fatty acids was made by comparing retention times of actual standards and by calculating, according to Ackman (1965), the relative retention times to oleic acid (18:1). For quantification the height (H) of each determined peak was measured to the nearest 1 mm, and the width at the half height (W~ 2H) was measured to the nearest 0.1 mm by the aid of a pair of vernier callipers. The area of each peak (H x W , ; n) and the percentage distribution were calculated without any correction factors. Minor components were excluded in the quantification. Hydrogenation of methyl esters After analysing the fatty acid composition of the methylated lipid extract from liver tissue some Platinum-IV oxide (Gelhard, Stockholm, Sweden) was added to the heptane phase containing the methyl esters. The tubes were then filled with hydrogen gas at a pressure of 2 atm and allowed to stand overnight. 1/*1 of the upper phase was injected for GLC. Identification and quantification were performed as above. Statistical treatment The data after 47, 96 and 164 days were tested against those after l 1 days and also against those from the preceding sampling occasion. All statistical tests were made by
Student's t-test. Significant differences were established at the 0-05-level. RESULTS AND DISCUSSION
The effects of starvation on muscle and liver triglyceride-phospholipid (TG-PL) ratio, phospholipid cholesterol ( P L - C H O L ) ratio, percentage of myristic acid (14:0) and on the percentage of liver total C~4fatty acids (after hydrogenation) are presented in Fig. 1. The effects on C16-, C,s-, C2o- and C2_~-fatty acids are presented in Figs. 2, 3, 4 and 5 respectively.
Triglycerides, phospholipids a~d cholesterol The absolute percental changes (w/w) in triglycerides, phospholipids and cholesterol are presented elsewhere (Dave et al., 1975). As the analysis of the fatty acid composition in this study was made on total lipid extracts, the relative proportions are more important than the absolute amounts of the different lipid classes. According to Addison et al. (1968) the fatty acid composition of the different lipid classes showed great differences in muscle from the cod, Gadus morhua. Triglycerides compared to the other lipid classes contained more 14:0, 16:1, 18:1, 20:1 and 22:1 and less 20:5 and 22:6. Muscle triglycerides also closely resembled the total lipids in liver as regards fatty acid composition. In a study on the Northern pike, Esox lucius L., Kluytmans & Zandee (1973b) obtained somewhat different results, but also these authors found that 18:1 was more abundant in the triglyceride fraction than in other lipid fractions including monoand diglycerides. The latter is in line with the observation by Brockerhoff & Hoyle (1963) that in fish oil 12 I
T G - P L r01"io (
4~ (H)
(~)
2/
i
r
(8)
P L - CHOL.rotio
h
Muscle Z*I4:0 (u) I
{8) I
Liver
--{ PL-CHOL rotio I
02)
(8)
~,~
~,2~z~----~ ....
"*TG-
PL
rQl"io
Liver I* (lO) ]
II
47
001 f 96
114:0 i
Liver 4 " 14 : tol-a I 164
Doys
Fig. 1. Effects of starvation on fatty acid composition in the eel. Triglyceride-phospholipid (TG PL) ratio, phospholipid-cholesterol (PL-CHOL) ratio in liver and muscle tissue. Myristic acid (14:0) in muscle and liver total lipids (%). Total C14-fatty acids (14:total) in liver lipids ('},% Results are expressed as mean +_ S.E. Number of fishes in parentheses; *P < 0.05 when tested against the results after 11 days; **P < 0'05 when tested against the results from the preceding sampling event.
Effects of starvation in the European eel triglycerides 18:1 is more abundant in the a-position than in the fl-position. In the latter study it was also e I ~l) found that fish triglycerides, like lecithins of most vertebrates, preferably store the polyunsaturated fatty acids in the//-position. The assumption has also been 20 ( ) (111) ~)16:O made that the polyunsaturated acids can be retained in the //-position in the lipid metabolism along the 12 I ~ Liver food chains by means of the chemical stability of the Io ( 16:1 fl-monoglyceride structure (Brockerhoff et al., 1963). In the present study the triglyceride-phospholipid (TG-PL) ratio was initially (11 days) about six times .Liver higher in muscle compared to liver (Fig. 1). As shown 26 ~ 16;O in a parallel study (Dave et al., 1975) the triglyceride content decreased more in muscle compared to liver, 24 ) but the decrease in T G - P L ratio in muscle between l 22 ~ (~o) 11 and 96 days was only 33% while the decrease in I (12) liver was more Ihan 50%. This more pronounced (12) 20 reduction in TG PL ratio in the liver during starvaI I tion was also accompanied by greater changes in fatty Liver acid composition in this tissue compared t6 muscle 30 16 : total (8) tissue. 1 28 The phospholipid-cholesterol (PL-CHOL) ratio (12) 1 I was of the same magnitude in liver and muscle. The 96 164 47 fluctuations in this ratio were almost parallel in liver Doys Fig. 2. Effects of starvation on fatty acid composition in and muscle during starvation (Fig. 1), For muscle the the eel. Palmitoleic acid (16:1) and palmitic acid (16:0) increase between 47 and 96 days was significant in muscle and liver total lipids (%). Total C16°fatty acids (P < 0-05). This effect might be due to changes in (16:total) in liver total lipids (%). environmental temperature. Thus the increased PLResults are expressed as mean + S,E. Number of fishes CHOL ratio between 47 and 96 days can he explained in parentheses; *P < 0.05 when tested against the results by the reduced muscle cholesterol content, which after 11 days; **P < 005 when tested against the results occurred contemporarily with a decline in water temfrom the preceding sampling event. perature (Dave et al., 1975). The change in muscle cholesterol content might be a result of an altered ~(12) (lit 3 ) Muscle
,of 02)
~ cult
Musc,e 16:1
I
361 I
t
51-
*~**
I
I Muscle
6~ (12)
(lit
(11)
(8) Muscle
[ (~)
?
~,~t
(8,)Musc,e
36
Liver
I
34
18:1
i| I 4~
I (tl)
I 20:4 Is) Muscle
3]-(ij2)~'''~ (~l)
(ll~l)
J
(8)]18:0
02)
(~) .....---[
~ 20:1
6t"30
4
28
26
)
I
6
I
~
I
(
4 [- ~
40 { 32 ~
) {'P)
18:0Liver
1121~
Liver
18 : total
56
32
I
I
96 Days
l
j-* Liver 20 : 5
)
I
*
4
61-~.'~
!,,2t
.Liver 20:4
~
)
2f ( 0 141]2f II
47
1-
o
~
,
[8)
,
~
,
Liver .20:1
*///._._~.~* //~c~
~Oi-Ozt/I / ~P~ t,2)
) I
~
~I-
' ~ [ - -
34
(
/ I
I 6
(8)
~
(
47
I
96
Liver (,) 20 : total I
164
Days
164
Fig. 3. Effects of starvation on fatty acid composition in the eel. Oleic acid (18:1) and stearic acid (18:0) in muscle and liver total lipids (%). Total Cla-fatty acids (18:total) in liver total lipids (%). Results are expressed as mean + S.E. Number of fishes in parentheses; *P < 0.05 when tested against the results after 11 days; **P < 0"05 when tested against the results from the preceding sampling event.
Fig. 4. Effects of starvation on fatty acid composition in the eeL Eicosapentaenoic acid (20:5), eicosatetraenoic acid (20:4) and ¢icosenoic 120:1) and/or linolenic acid 08:3) in muscle and liver total lipids (%). Total C20-fatty acids (20:total) in liver total ]ipids (%). Results expressed as mean _+ S.E. Number of fishes in parentheses; *P < 0.05 when tested against the results after 11 days; **P < 0-05 when tested against the results from the preceding sampling event.
512
GORAN DAVE et al.
I 5 L| (~_ 3 '
1,0 I (u) ] f
~
Mu,c,e
I
I~l 22 : 5 I
m) I I
(s) Muscle ! 22:4
161 ~ 1 4
~(8)
Liver 22:5
12 (i2) i0
F'~
!
(iIo)
I
~
24
Liver 22:4
(
) I
I
I Liver 22: totol
~ *
22
20
/
(~)
16 ( 14 1 III
Oz) 47I
1 96 Days
I 1£;4
Fig. 5. Effects of starvation on fatty acid composition in the eel. Docosapentaenoid acid (22:5} and docosatetraenoic acid (22:4) in muscle and liver total lipids (.,,). Total Ce2-fatty acids (22:totalt in liver total lipids (%{i)Results are expressed as mean Jr S.E. Number of fishes in parentheses; *P < 0.05 when tested against the results after 11 days; **P < 0.05 when tested against the results • from the preceding sampling event. activity in plasma lecithin-cholesterol acyltransferace (LCAT). This role of LCAT, i.e. performing changes in cholesterol content of biological membranes and thus changing their properties, is suggested to be its main physiological role in mammals (Glomset, 1970). The present results suggest that a similar role of LCAT can be valid also in fish. 14:0 and 14: total.latty acids
The decrease in 14:0 in both liver and muscle (Fig. 1) was almost parallel with the decrease in T G PL ratio (except for muscle after 164 days). This is in line with the finding of Addison et al. (1968) that 14:0 is found mainly in the triglyceride fraction• The values for 14: total in liver tissue showed a successive decrease during the starvation period. When comparing the present results on liver 14:0 and liver 14: total (after hydrogenation) it is observed that the values of 14:0 exceeded those of 14: total. This is theoretically impossible and must be due to a more accentuated overestimation of earlier peaks compared to later peaks in the GLC analytical procedure for the non-hydrogenated than for the hydrogenated samples. In the former the polyunsaturated fatty acids are represented by later and broader peaks. Furthermore, myristoleic acid (14:1), which only constituted a minor component in the eel fat, was not quantified at any occasion as no detectable changes in this fatty acid were observed. Accordingly, 14:1 is not likely to contribute to any observed change in 14: total and therefore the latter parameter can be regarded as a
more accurate estimation of the content of 14:0 in liver tissue. 16:0. 16:1 and 16 : total fittly acids
In muscle lipids from the cod 16:1 had a clear preference for the triglyceride fraction, while 16:0 had a more complex distribution in different lipid classes (Brockerhoff et al., 1963). In fish triglycerides neither 16:0 nor 16:1 fatty acids did not appear to show any particular preference for :~- or fl-position (Brockerhoff & Hoyle, 1963), while in liver and muscle phosphatidyl choline of the cod both 16:(t and 16:l showcd a marked preference for the >position (Brockerhoff et al.. 1963). In the present study the starvation did not causc any changes either in muscle 16:0 and 16:1. or in liver 16: total thtty acids. On the other hand, there was a continuous decrease in liver 16:1 and a parallel increase in 16:0 (Fig. 2). This combined effect of starvation on liver 16: fatty acids is hard to explain on the basis of different distribution in lipid classes or of preferential ~- or fi-positions. Thc effect is therefore more likely to be seen as a reduced olefin synthesis ability in the starving animal. Such an assumption is supported by results obtained in studies on fasting rats (Elovson, 1965; Benjamin & Gellhorn, 1966: kec & Sprecher. 1971). 1~:0, 18:1 and 18: total/arty acids
According to Addison et al. (1968) 18:0 showed a relatively even distribution among most of the different lipid classes in cod muscle. One exception was serine phosphoglyceride, which had a content o1" 18:0 3 6 times higher than the other lipid classes. 18:t. on the other hand, showed some preference for the triglyceride fraction. The latter finding has also been observed in the Northern pike (Kluytmans & Zandee, 1973b). These authors observed that the triglyeeride fraction was relatively poor in IS:0. Initially (11 daysl the amounts of both 18:0 and 18:1, expressed as percentage of total estimated fatty acids, were equal in liver and muscle tissue. However. the individual variations, indicated by the standard errors (S.E.), were more pronotinced in liver compared to muscle tissue lFig. 3). The levels of 18:0 and 1~:1 in lnuscle tissue were relatively unaffected during starvation except for the slight increase in 18:0 between 11 and 47 days. The liver, on the other hand, showed a pronounced decrease in 18:1 and 18: total, while the level of 18:0 was almost unchanged. These findings, together with other observations, suggest: 1. A pronounced difference in lipid metabolism in muscle and liver. 2. A decreased olefin synthesis in the liver ot the starving eel. 3. Compared to other fatty acids, 18:1 appears to provide the most energy for metabolic purposes in lasting eels. The first suggestion is based on the fact that the levels of 1g:0 as well as 18:1 were equal in liver and muscle initially (11 days), but then the utilization of preferential fatty acids, like 18:1, is much more pronounced in the liver although the lipid class utilized mainly in both organs is triglycerides (Fig. l j. The
Effects of starvation in the European eel second suggestion agrees with similar findings for 16:1 in the present study and is further supported by results obtained in fasting mammals (Elofson, 1965; Benjamin & Gellhorn, 1966; Lee & Sprecher, 1971; Seedy & Poole, 1974). The third suggestion is based on the findings that the decrease in liver 18:1 was not accompanied by any comparable increase in 18:0. Instead the liver 18: total content diminished. Furthermore, the increase in liver 16:0 cannot explain the decrease in both 16:1 and 18:1. Thus, the reduced liver 18:1 cannot solely be due to a decreased olefin synthesis. The assumption that the primarily utilized fatty acid was 18:1 is also in agreement with the observation that 18:1 seemed to be the most preferably located fatty acid in the ~t-position in the liver triglycerides of the American eel, Anouilla rostrata (Brockerhoff & Hoyle, 1963). Therefore 18:1 is, to a higher degree than the other fatty acids, likely to be hydrolysed from the triglyceride molecule and enter the d-oxidation for energy purposes. Probably this is valid also for other vertebrates. Thus, it has been reported that 18:1 was the fatty acid showing the greatest decrease in carcass fat during starvation in the neonatal swine (Seedy & Poole, 1974). 20:1 and/or 18:3, 20:4, 20:5 and 20: total fatty acids In the muscle lipids of cod, Addison et al. (1968) found that 20:1 was about three times more abundant in triglycerides than in other lipid classes. In addition 18:3 and 20:4 showed no obvious preference for any special lipid class, while 20:5 was two to three times more abundant in sterol esters and choline phosphatides than in other lipid classes. Kluytmans & Zandee (1973b) observed preference of all these fatty acids in triglycerides compared to phospholipids in the Northern pike. In the triglycerides of the American eel, Anouilla rostrata, 20:1 showed preference for the a-position, while 20:5 together with 20:3 and/or 20:4 showed preference for the d-position (Brockerhoff & Hoyle, 1963). A similar preferential distribution was also observed in phosphatidyl choline of cod liver and muscle, as well as in muscle of the scallop, Plactopecten ma#ellanicus, and liver of the lobster, Homarus americanus (Brockerhoff et al., 1963). In the present study no significant changes were observed in muscle C2o fatty acids (Fig. 4) in spite of changes in the T G - P L ratio. In the liver, on the other hand, there was a marked increase in 20:4 and 20:5 and a marked decrease in 20:1. 20: total fatty acids increased in the liver during starvation. The changes in C2o fatty acids of the liver can certainly be explained by various distributional patterns of these fatty acids in the different lipid classes and ~or d-positional preference as described above. The disagreement between changes in C2o-fatty acids in" liver and muscle might therefore be due to different regulation of the lipid metabolism in liver and muscle. In the eel the main part of the depot fat is stored in the muscle (Dave et al., 1975), which showed very limited alterations in the fatty acid pattern. Accordingly there is no profound change in fatty acid pattern of the whole fish. Therefore deficiency symptoms caused by lack of polyunsaturated fatty acids are not likely to occur during starvation in the eel. Deficiency symptoms like impairment of growth and
513
pigmentation have previously been reported in fry of chinook salmon, Oncorhynchus tshawytscha, fed a fatfree diet for 24 weeks (Nicolaides & Woodall, 1962). 22:4, 22:5 and 22: total fatty acids The identification of polyunsaturated fatty acids, generally occurring in fish lipids, by means of the retention time after GLC analysis, is mostly unprecise, especially regarding the degree of unsaturation. Comparisons between different studies should therefore be made with great caution. In the present study no efforts were made to identify the fatty acids by other means than GLC before and after complete hydrogenation. Accordingly only the number of carbon atoms is determined more precisely while the number of double bonds is more uncertain. The starvation did not cause any significant alteration in the content of 22:4 and 22:5 in muscle tissue (Fig. 5). On the other hand, in liver tissue 22:4 increased during the first 96 days, while 22:5 and 22: total showed a successive elevation during the progress of the starvation. With reservation for the difficulties mentioned above, it is yet likely that the fatty acids identified in the present study as 22:4 and 22:5 are the same as those identified by other authors (Addison et al., 1968) as 22:5 and 22:6 respectively. These two acids have been found to be mainly located in non-triglycerides of cod muscle lipids (Addison et al., 1968). In the triglycerides of the American eel, Anguilla rostrata, they were further found to be preferably located in the d-position (Brockerhoff & Hoyle, 1963). Such reasons are probably responsible for the percental increase in 22:4 and 22:5 in the liver, as the phospholipids are regarded as structural elements in contrast to triglycerides, and the fatty acids in the d-position are not as easily hydrolysed as those in the or-position.
CONCLUSIONS
Starvation in the European eel, Anguilla anguilla, has a pronounced influence on the fatty acid composition of the liver. The muscle, which is the main fat depot in the eel, on the other hand, shows much smaller alterations in fatty acid composition. The changes in fatty acid composition can be partly explained by altered ratios between the different lipid classes, which in other aquatic animals have been shown to have differences in fatty acid pattern (Addison et al., 1968; Kluytmans & Zandee, 1973b). Also preferential location in ~- or fl-position in triglycerides and lecithins (Brockerhoff et al., 1963; Brockerhoff& Hoyle, 1963) might contribute to some of the observed changes. These reasons might be valid for the continuous decrease in 14:0 in both liver and muscle and the increase in liver polyunsaturated acids (20:4, 20:5, 22:4 and 22:5). Disagreement of the starvation-induced changes in the fatty acid pattern between liver and muscle suggests a different regulation of the lipid metabolism in these two tissues. The changes in the monoenes (16:1, 18:1 and 20:1 and/or 18:3) in liver tissue are in line with findings on mammals that olefin synthesis is almost completely abolished during starvation. Studies on rats (Elovson, 1965) have further shown
514
GORAN DAWz et al.
that in extrahepatic tissues desaturation a n d elongation of fatty acids are less p r o m i n e n t than in liver. The more p r o n o u n c e d changes in liver c o m p a r e d to muscle might be a n indication that also in the eel the liver is the main organ for fatty acid metabolism (synthesis, interconversion a n d oxidation). The transient increase in muscle p h o s p h o l i p i d cholesterol ratio observed at 96 days (Fig. 1) might be a response to the lower water temperature by this time. Thus, the lowered muscle cholesterol content after 96 days would, as cholesterol is considered as mainly a m e m b r a n e constituent, adjust the fluidity of the membranes. This m e c h a n i s m of adjusting the m e m b r a n e properties in relation to temperature seems more probable than alterations in the proportions of saturated a n d polyunsaturated fatty acids. The changes in fatty acid pattern of the liver lipids are very similar to observations made during cold acclimation, i.e. an increase in polyunsaturated fatty acids. However, the only fatty acid showing some relation to water temperature (presented by Dave et al., 1975) was 22:4, while the other polyunsaturated fatty acids (20:4, 20:5 a n d 22:5) did not. In studies on temperature influence on the fatty acid composition in fish the nutritional state must therefore be considered carefully, as m a n y fish species, including the eel, lose appetite when the temperature is lowered. The percental a m o u n t s of myristic acid (14:0), palmitoleic acid (16:1) a n d oleic acid (18:1) in liver lipids decline almost continuously during starvation and might serve as an indication of the nutritional state in the eel a n d possibly also in other tish species. Acknowled,qements The authors wish to thank Mrs. Sir ()stling for her excellent technical assistance during this work. We also wish to thank Dr. Bertil Swedmark at Kristineberg Zoological Station. The financial support of the National Board of Fisheries, Sweden, is also gratefully acknowledged. REFERENCES
ACKMAN R. G. (1965) Cod liver oil as secondary standards in the GLC of polyunsaturated fatty acids of animal origin: analysis of a dermal oil of the Atlantic leatherback turtle. J. Am. Oil Chem. Soc. 42, 38 42. ACKMAN R. G. (1967) Characteristics of the fatty acid composition and biochemistry of some fresh-water fish oils and lipids in comparison with marine oils and lipids. Comp. Biochem. Physiol. 22, 907 922. ADDISON R. F., ACKMAN R. G. & HINGLEY J. (1968) Distribution of fatty acids in cod flesh lipids. J. Fish Res. Bd Can. 25, 2083--2090. ARM S., NOSE T. & HASHImOtO Y. (1971) A purified test diet for the eel, Anguilla japonica. Bull. Freshwater Fish. Res. Lab. 21, 161 178. BENJAMIN W. & GELLHORN A. (1966) RNA biosynthesis in adipose tissue: effect of fasting. J. Lipid Res. 7, 285294. BROCKERHOFF H., ACKMAN R. G. & HOYLE R. J. (1963) Specific distribution of fatty acids in marine lipids. Arch. Bioehem. Biophys. 100, 9 12. BrOCKERHOFF H. & HOYLE R. J. I1963) On the structure of the depot fats of marine fish and mammals. Arch Biochem. Biophys. 102, 452-455. CALDWELL R. S. & VERNBER6 J. F. (1970) The influence of acclimation temperature on the lipid composition of fish gill mitochondria. Comp. Bioehem. Physiol. 34. 17% 191.
CARLSSON L. A. (1963) Determination of serum triglycerides. J. Atheroscler. Res. 3, 334~336. DAVE G., JOHANSSON M.-L., LARSSON A., LEWANDER K. & LIDMAN U. (1974) Metabolic and hematological studies on the yellow ane silver phases of the European eel, 4nguilla anyuilla L. II. Fatty acid composition. Comp. Biochem. Physiol. 47B, 583 591. DAVI! G., JOHANSSON-Sf)BECK M.-L., LARSSON /~.. LEWANDI~P,K. & LIDMAN U. {1975) Metabolic and hematological effects of starvation in the European eel, Amduilla amluilla L. h Carbohydrate, lipid, protein and inorganic ion metabolism. Comp. Biochem. Physiol. 52A, 423 430. ELovsox J. (19651 Conversions of palmitic and stearic acid in the intact rat. Biochim. hiophys. Acta 106, 291 303. FAr~ZAST. & HErODEC S. (1964) The effect of environmental temperature on the fatty acid composition of crustacean plankton, J. Lipid Res. 5. 369 373, GLOMS~iTJ. A. (1971)) Physiological role of lecithin cholesterol acyltransferase. Anl..I. clin. Chem. 23, 1129-1136. HAZH, J. R. & ProssEr C. L. (1974} Molecular mechanisms of temperature compensation in poikilotherms. Physiol. Ret..54 {3), 596619. HOAR W. S. & Cor'rLE M. K. (1952) Some effects of temperature acclimatization on the chemical constitution of goldfish tissues. Can. J. Zool. 30, 49 54. JOHANSSON-SJI~BI!CK M.-L., DAVE G., LARSSON ,~., L1WANDIR K. & LIDMAN U. 11975) Metabolic and hematological effects of starvation in the European eel. Anguilla an¢luilla L. II. Hematologs,. Comp. Biochem. Physiol. 52A. 431 434. JoH~,srox P. V. & Roots B. 1. (1964) Brain lipid lhtt2r acids and temperature acclimation. Comp. Biochem. Physiol. II. 303 309. KANI!KO T., TAKI-UCHI M,. ISHll S., HIGASHI H. 8¢~ KIKUCHI T. (1966) Effect of dietary lipids on fish under cultivation IV. Changes of fatty acid composition in flesh lipids of rainbow trout on nun-feeding. Bull. Jap. Soc. Sciem. F'ish. 33, 56 58 (abstract in English). KtMp P. & SrvHt;t M. W. [1970) Effect of temperature acclimatization on the fatty acid composition of goldfish intestinal lipids. Bioehem. J. 117, 9 15. KLt;;'TMANS J. H. F. M. & ZaYDtl: D. 1. (1973a) Lipid metabolism in the Northcrn pike (Esox lucius L.} -I. The fatty acid composition of the Northern pike. Comp. Biochem. Physiol. 44B, 451 458. KLUYTMANS J. H. F. M. & ZANDEE D. J. 11973b) Lipid metabolism in the Northern pike (Esox lucius k.) II. The composition of the total lipids and of the fatty acids isolated from lipid classes and some tissues of the Northern pike. Comp. Biochem. Physiol. 44B, 459 466. KNU'PRATtt W. G. & MEAD J. F. (1967) The effect of environmental temperature on the fatty acid composition and on the i~ vivo incorporation of [1-~4C]-acetate in goldfish (Carassius auratus L.). Lipids 3. 121 128. LEk C.-J. & SP~¢EC'HERH. 11971) An in vitro study of the efl'ects of dietary alterations and fasting on the desaturation of palmitic, stearic eicosa-8,11-dienoic and eicosa8,11,14-trienoic acids. Bioehim. hiophys. Acta 248, 180 185. LOVE R. M. (1970) The Chemical Biolo~ty o/ Fishes, pp. 216 217. Academic Press, New York. Lovers J. A. (1938) Fat metabolism in fishes--Xlll. Factors influencing the composition of the depot fat o[" fishes. Biochem. J. 32, 1214-1224. LOW:RN J. A. (1940) Fat metabolism in fishes. The utilization of the ethyl esters of fatty acids by the eel and their effect on depot fat composition. Biochem. J. 34, 70,-~708. LOVI-:RN J. A. (1950l Some causes o[" wmation in the composition of fish oils. J. Soc. Leath. 7i'ades Chem. 34, 7 21. NICOLAn)ES N. & WoOI)AEl A. N. {1962) lmpaired pig-
Effects of starvation in the European eel mentation in chinook salmon fed diets deficient in essential fatty acids. J. Nutr. 78, 431-437. REISER R., STEVENSONB., KAYAMAM., CHOUDHURYR. B. R. & HOOo D. W. (1963) The influence of dietary fatty acids and environmental temperature on the fatty acid composition of teleost fish. J. Am. Oil Chem. Soc. 40, 507-513. ROOTSB. J. (1968) Phospholipids of goldfish (Carassius auratus L.) brain: the influence of environmental temperature. Comp. Biochem. Physiol. 25, 457-466.
515
ROOTS B. J. & JOHNSTONP. V. (1968) Plasmalogens of the nervous system and environmental temperature. Comp. Biochem. Physiol. 26, 553-560. SEERLEYR. W. • POOLE D. R. (1974) Effect of prolonged fasting on carcass composition and blood fatty acids and glucose of neonatal swine. J. Nutr. 104, 210-217. TASHIMAL. & CAHILLG. F. (1965) Fat metabolism in fish. In Handbook of Physiology (Edited by RENOLD A. E. & CAHILLG. F. JR.), Sect. 5, Adipose tissue, pp. 5~58. American Physiological Society, Washington, D.C.
