Metabolism of fatty acids in fish

Metabolism of fatty acids in fish

Aquaculture, 20 (1980) 29-40 o Elsevier Scientific Publishing 29 Company, Amsterdam - Printed in The Netherlands METABOLISM OF FATTY ACIDS IN FIS...

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Aquaculture, 20 (1980) 29-40 o Elsevier Scientific Publishing

29 Company,

Amsterdam

- Printed

in The Netherlands

METABOLISM OF FATTY ACIDS IN FISH III. COMBINED EFFECT OF ENVIRONMENTAL TEMPERATURE AND DIET ON FORMATION AND DEPOSITION OF FATTY ACIDS IN THE CARP, ClYPRINUS CARP10 LINNAEUS 1758

TIBOR FARKAS*,

ISTVAN

CSENGERI,

FERENC

MAJOROS

and JiNOS

OLAH

Fisheries Research Institute, H-5541 Szarvas (Hungary) * Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged (Hungary) (Accepted

10 June 1979)

ABSTRACT Farkas, T., Csengeri, I., Majoros, F. and OIBh, J., 1980. Metabolism of fatty acids in fish. III. Combined effect of environmental temperature and diet on formation and deposition of fatty acids in the carp, Cyprinus carpio Linnaeus 1758. Aquaculture, 20 : 29-40. Formation and deposition of fatty acids in carp maintained on diets differing in total fat as well as in linolenic acid content was investigated by following the incorporation of (1-‘%)-acetate into liver total- and phospholipid fatty acids at two extreme temperatures (5” and 25” C). Excess dietary linolenic acid was deposited in triglycerides but not in phospholipids. The formation and level of phosphoIipid docosahexenoic acid was, however, dependent on the amount of linolenic acid in the diet. Despite the vast quantities of ingested linolenic acid, the carp on diets containing sufficient essential fatty acid maintained similar membrane fluidities as judged from the ratio of saturated to unsaturated fatty acids. Decrease of the environmental temperature brought about a reduction in the rate of formation of palmitic acid and an increase in the rate of formation of docosahexenoic acid in carp receiving sufficient essential fatty acid. Consequently, the level of palmitic acid decreased and that of docosahexenoic acid increased in the liver phospholipids in carp and a number of other fish species. Essential fatty acid deficient carp were unable to increase the rate of production of long chain polyunsaturated fatty acids upon exposure to cold, The results are discussed from the point of view of adaptation of membrane fluidity to the temperature by fish, and the importance of docosahexenoic acid in this process is emphasized.

INTRODUCTION

Environmental temperature and diet can be specified as the factors exerting major impact on the fatty acid metabolism and fatty acid composition of fish. Available data show that the biosynthesis of fatty acids depends on the type of food fed to fish (Farkas et al., 1978) and that the fatty acid composition of deposited as well as of structural lipids is influenced by

30

dietary fatty acids (Reiser et al., 1963; Stickney and Andrews, 1971; Caste11 et al., 1972; Worthington and Lowell, 1973; Watanabe et al., 1975; Yu and Sinnhuber, 1976). Environmental temperature has also been shown to govern fatty acid composition (Hazel and F’rosser, 1974) and ability to adapt to temperature can be regarded as a survival factor for poikilothermic animals, including fish. The effects of these factors were studied in separate experiments, i.e. fish were fed at constant temperature with various diets, and temperature effects were studied on fish maintained on standard diets. Under natural conditions, however, there is no clear-cut situation as these factors act simultaneously on fatty acid metabolism of fish. Thus, the observed fatty acid composition is always the resultant of these two vectors. Carp (Farkas and Csengeri, 1976) along with other poikilothermic aquatic animals (Farkas 1971,1979) were shown to adapt their fatty acid metabolism to the prevailing temperature rather rapidly under laboratory conditions but we have no information on how this response can be modified by dietary means. To find the conditions under which fish give maximal response to decrease of temperature would also be of economic significance, as such fish would probably be more vital at reduced temperatures, and would be more successful in surviving the low temperature period. In this paper the effect of environmental temperature on fatty acid biosynthesis of fish held on different diets is investigated. Data are also presented, showing immediate changes in phospholipid fatty acid composition of fish exposed to cold. MATERIAL

AND METHODS

Animals Carp weighing 15-25 g were fed in the laboratory for 4-10 weeks on feeds differing in fat content and fatty acid composition. Information about fat content and fatty acid composition of the diets used is given in Table I. Carp (Cyprinus carpio L.), sheatfish (Silurus glanis L.), bighead carp (Hypophthalmichthys nobilis R.), and silver carp (Hypophthalmichthys molitrix Val.) weighing 400-600 g were collected from their natural habitats around the institute in the summer of 1977. Labeling of animals in vivo Five animals in each group were injected abdominally with sodium (l-14C) -acetate (spec. act. 17 mCi/mM, 0.1 pmole/g fish) at selected intervals before sacrifice. Some fish were exposed to 5” C while others remained at 25°C after injection. Incorporation of radioactivity into liver fatty acids was linear with time for at least 4 h in fish at 25°C and for 8 h in fish exposed to 5°C. At the end of incubation the livers were removed from freshly killed animals into ice-cold saline containing 0.1 M cold sodium acetate. After thorough rinsing, pieces of liver were homogenized.

31 TABLE I Fat content and fatty acid composition of the diets Diet no. Fat content (%)

I

II 3.4

III

1.7

Fatty acid

weight percentage

14:o 16:0 18:0 18:l 18:2 18:3

0.33 18.41 2.16 26.31 47.51 1.50

3.05 25.33 7.67 37.22 24.22 2.84

3.7

1.12 14.43 5.79 27.77 33.11 15.04

IV

V

10.4

20.0

1.06 9.64 3.90 21.88 35.13 22.35

0.83 9.64 4.60 23.83 32.88 28.28

Only the major fatty acids are listed and so the sum of fatty acids does not necessary equal 100%.

Extraction and separation of lipids Details of lipid extraction and separation have been described in earlier papers (Farkas and Csengeri, 1976; Farkas et al., 1977; Farkas et al., 1978). Briefly, 1:ipids were extracted according to the Folch technique (Folch et al., 1957) and separation of lipid classes was performed on silicagel G plates using the solvent system petrol ether : ether : acetic acid = 85 : 15 : 1. In some cases phospholipid from the start and triglyceride were removed in ampoules containing 5% HCl in absolute methanol and transesterified at 80°C under an inert atmosphere. In other cases the total lipid fraction was transesterified with the above technique. GLC separation of fatty acids was performed using a JEOL JGC 1100 apparatus. Most of the probes were separated on 11% EGSS-Y on 100-120 mesh Gas-Chrom P in a 6-foot-long coiled glass column at 175°C. In certain cases 15%) DEGS on 100-120 mesh Gas-Chrom P was used to separate the methyl es’ters. This column was programmed from 130’ to 175°C to give a partial separation of 18 : 3~3 and 20 : 1~9. To collect the radioactivity, the columns were split before their outlet in a ratio of 1 : 10. Collection of radioactivity took place in glass cartridges as described earlier (Farkas and Csengeri, 1976). Triangulation technique was employed to calculate the percentage distribution of fatty acids. RESULTS

Effect of diet on fatty acid composition The amount

and proportion

of both linoleic and linolenic

acid in the diet

32 TABLE II Triglyceride fatty acid compositions Diet no.

I

II

Fatty acid

weight percentage

14:o 16:0 16:l 18:O 18:l 18:2 18:3

0.95 15.23 7.52 4.69 55.86 7.78 2.43

2.07 19.88 13.25 4.26 49.15 6.15 0.46

of carp on different diets III

IV

V

1.98 18.05 9.23 3.71 43.03 12.98 4.95

3.34 15.54 5.84 3.75 43.38 17.52 7.51

1.15 9.84 4.66 2.05 25.03 29.63 19.86

Only the major fatty acids are listed.

TABLE III Phospholipid datty acid compositions Diet no.

I

Fatty acid

weight percentage

16:0 16:l 18:0 18:lw9 18:2w6 18:3w6 18:3w3 2O:lw9 20:2w6 20:3w9 20:3w6 20~4~6 20:4w 3 20:5w 3 22:4w6 22:5w6 22:5w 3 22~6~ 3

21.06 4.42 7.63 17.11 0.91 0.48 0.95 7.19 2.17 19.84 0.26 0.30 1.43 6.07 0.52 8.88

20:3w9

22~6~3 B

sat.

Z unsat.

II

27.15 7.64 4.07 21.38 5.70 -

-

-

1.83 1.70 4.24 1.22 8.08

0.37 0.32 3.22 0.42 10.08

0.80

0.42

0.40

0.45

of carp on different diets III

IV

V

27.84 4.26 10.58 11.79 5.18 0.43 1.00 2.00 1.21 -

27.91 3.00 11.03 11.93 6.15 1.12 1.42 0.82 -

31.56 3.74 5.68 9.75 11.57 0.14 2.95 1.22 0.86 -

1.42 9.80

1.95 9.11

0.93 4.38

-

-

-

0.71 0.92 1.38 0.35 19.92

1.05 0.45 0.30 0.71 22.43

2.59 0.36 0.14 0.79 22.72

-

-

-

0.62

0.63

0.59

33

appear to govern the fatty acid composition of liver triglycerides. With increasing amounts of ingested linoleic and linolenic acids, the level of oleic acid decreased, and parallel to it, that of linoleic and linolenic acids increased (Table II). The same is true for phospholipids as well, although the level of linolenic: acid remained at low levels in all fish. Fish on diet I represent an exception: in spite of the high dietary level of linoleic acid, this fatty acid remained at low levels in both phospholipids and triglycerides. Phospholipid arachidonic acid was inversely related to the linoleic acid content of the diet (Table III). The most spectacular change occurred with phospholipid docosahexen’oic acid: its level rose abruptly from 8-10% in fish fed with diets I and II to 20% in fish ingesting diets III, IV and V. Fish receiving the former diets exhibited a relatively high ratio of 20 : 3~9 to 22 : 6~3 (0.80 and 0.40, respectively). Effect o,f temperature on fatty acid biosynthesis In table IV the percentage distribution of radioactivity of fish adapted to warm coaditions and of those exposed to 5°C for 6 h is compared. The control fish were injected with sodium (l-14C)-acetate 1 h before sacrifice. Comparing the radioactivities at the times of equal levels of tissue labeling would have been more correct. Equal level of tissue labeling in we-adapts fish was at 30-40 min; thus we feel that the use of fish allowed to incorporate radioactivity for 1 h does not cause misleading results. From Table IV it is evident that the major product of fatty acid biosynthesis in warm injected animals was palmitic acid, ~omp~ing about 30-40% of the incorporated radioactivity. Two exceptions can be observed however. In fish fed diets I and V, only one third of this value appeared in the palmitic acid. Oleic acid picked up a considerable proportion of radioactivity also, indicating its intensive formation at 25°C. The radioactivity appearing in the total long chain polyunsaturated fraction did not s’eem to be a function of the amount and type of unsaturated fatty acid in the diets. The degree of labeling remained between 10 and 20% with increasing fat content of the diets even in fish on a diet containing 20% fat. On the other hand, formation of docosahexenoic acid was dependent on the level of linolenate in the diet. Table IV shows that all fish reorganized the pattern of fatty acid biosynthesis upon exposure to cold. In every case, a drastic reduction in labeling of saturated fatty acids followed the temperature shift. Concomitt~tly, a higher proportion of label was directed into the polyunsaturated fatty acid fraction. The overall result of these changes in fatty acid biosynthesis was that the fish exposed to cold produced a fatty acid profile which was more unsaturalt~ than in those exposed to warm water. This is evident also when we calcul.ate the ratio of labeling of total saturated to total unsaturated fatty acids. The type of ingested fatty acids exerted a profound effect on the intensity oE the response to the temperature shift. The fish receiving diet I and

2.2 3.3 1.3 1.4 2.2

2.0 2.8 2.8

4.0 1.1 1.2 1.9

2.2 2.3 3.0

0.40

20:3w6 20:4w6 20~4~3 20:5w3 22:4w6

22~5~6 22~5~ 3 22~6~3

cpm total saturated* cpm total unsaturated

-

-

-

0.95

1.0 1.6 1.4

1.0

0.5 0.2

2.9

-

0.24

5.8 4.6 1.9

4.6 2.6 5.8 2.6

1.5 3.1

-

2.48

0.1 0.5 1.3

0.1 1.3 1.0 1.8

0.20

3.6 5.6 10.9

-

3.0 3.4 3.2 3.8

5.2,

-

1.2

-

12.3 3.4 4.8 12.1 1.2 -

5

31.1 1.8 33.6 11.3 -

25

III

-

1.28

4.0 5.1

0.20

1.9 16.2

-

2.6 2.6 5.2 1.4

1.6 0.8 0.1 -

8.1 4.0 8.3 10.0 0.4 1.3 9.6 10.0 -

5

41.9 2.8 14.3 15.4 1.2 2.1 -

25

IV

-

0.16

4.0 6.2

4.1 1.8 8.6 3.3

9.1 4.2 4.4 4.4 3.1 6.2 6.8 10.2 -

25

V

0.05

9.0 1.5

8.5

8.0 5.8 10.5 6.8

4.0 2.2 1.5 1.1 5.6 4.9 10.9 9.3 -

5

Fish at 25°C were injected with (l-l%) sodium acetate 1 h, and those at 5°C 6 h before sacrifice. AI1 the fatty acids emerging from the column between 14:0 and 22:6 were collected and counted but only the most important of them are listed in the table. * These caclulations are based on the total fatty acid labeling data.

0.50

3.2 2.6

2.8 1.5

20:2w6 +9 20:3w9

-

10.8

3.4

20.0 1.6 28.8 9.3 9.3 2.2 2.0

6.9 2.6 12.1 11.6 6.3 1.6 9.0

9.2 2.2 29.1 12.6 2.5

20.4 1.3 8.6 26.3 3.5

16:0 16:l 18:O 18:l 18:2 18~3~3 2O:lw9

5

percentage distribution of radioactivity

25

Fatty acid

5

25

Temperature (“C)

II

I

Diet no.

Effect of temperature on fatty acid biosynthesis by carp held on different diets

TABLE IV

K

35

exhibiting a high ratio of 20:3w9 to 22:60 3 were unable to adjust the composition of the newly formed fatty acids to the temperature. In another experiment with essential fatty acid deficient carp the same result was obtained (data not presented). Decrease of labeling of palmitic acid was accompanied by increased formation of stearic acid in these cases. On the other hand, over 50% of the incorporated radioactivity was recovered from the polyunsaturated fatty acid fraction of fish ingesting a higher level of dietary fat (diets IV and V). The labeling of docosahexenoic acid increased from fish on diet II to diet IV. The response observed in fish on diet V was less intense but still pronounced. The observed increase in labeling of polyunsaturated fatty acids in cold is not the result of a decreased rate of labeling of palmitic acid. In some experiments the specific radioactivities of certain fatty acids were compared at the time of equal level of tissue labeling. The values for 16:0,18:0, 20:4 and 22:6 in the case of fish on diet III were 0.28, 0.98, 2.35 and 9.86, respectively. As will be explained in a forthcoming paper, values less than unity indicate a decreased and those higher than unity an increased rate of formation of the respective fatty acid. Thus exposure to cold of carp receiving sufficient essential fatty acid results in a stimulation of long chain polyunsaturated fatty acids. Effect of temperature shift on liver fatty acid compositions The increased rate of formation of polyunsaturated fatty acids in fish exposed to cold might bring about accumulation of these fatty acids and, conversely, decreased formation of saturated fatty acids can be expected to result in a diminished level of these fatty acids in different tissues. As a matter of fact, this was the response observed by several authors in fish exposed to cold for a long period of time (Knipprath and Mead, 1966a,b; Patton, 1.975; Farkas and Csengeri, 1976; Miller et al., 1976; Cossins, 1977; Cossins a.nd Prosser, 1978). The data in Tables V and VI demonstrate that adaptation of fatty acid composition to cold is more rapid than could be expected on the basis of the available information. Table V lists the fatty acid compositions of liver total lipids of warm- and cold-exposed fish while Table VI shows the effect of cold exposure on liver phospholipid fatty acid compositions of several fish species. These are the first data to show that fish rapidly and efficiently adjust the liver fatty acid composition to the prevailing temperature. Actually, a few hours of cold exposure was sufficient to elevate the level of long chain polyunsaturated fatty acids in total lipids as well as in phospholipids. The extent of the response is, however, dependent on the type of diet and also on the initial fatty acid composition of phospholipids. The fish on diet I failed to alter either the total lipid or phospholipid fatty acid compositions as they were also unable to increase the rate of formation of polyunsaturated fatty acids in the cold. On the contrary, fish consuming a diet with a medium level

-

0.12 0.71 1.48 0.15 1.42

0.47 2.87 0.71 4.00

1.07 16.34 6.75 6.16 50.44 5.03 0.24 1.30 -

14:o 16:0 16:l 18:O 18:l 18:2 18:3 18:3 2O:l 20:2 20:3 20:3 20~4 20:4 20:5 22~4 22:5 22:5 22~6

1.96 0.83 4.32 0.10 0.17 0.46 1.00 0.27 1.75

1.00 15.71 4.91 9.20 40.00 12.45 0.51 3.58 -

weight percentage

5

Fatty acid

25

(“C)

Temperature

2.71

0.95

1.48 21.82 12.35 5.07 43.38 2.16 0.25 0.46 2.47 0.30 1.56 0.34 3.68 -

25

II

1.41 19.41 10.53 4.41 34.22 3.94 0.18 0.56 1.97 0.18 4.32 0.37 7.61 0.28 0.28 0.37 2.82 0.47 6.06

5

shift on liver total fatty acid compositions

I

of temperature

V

Diet no.

Effect

TABLE

-

-

-

4.03

0.62

-

0.52 0.45 9.46

0.34 1.60 0.21 0.32

0.64 2.76

-

2.55 1.48 0.49

1.68 12.36 4.29 4.00 45.36 12.32 0.66 4.05 3.22 0.08 -

5

1.56 21.32 7.43 5.66 41.16 10.61

-

25

III

-

-

-

0.22 3.83

0.79 2.02

8.14 2.25 0.60

1.01 17.55 4.65 4.13 36.08 17.62 -

25

IV

-

0.07 0.27 2.45

0.44 1.24 0.13 0.65

0.89 13.70 3.46 3.11 35.44 20.02 0.36 11.38 2.94 2.07 -

5

0.12 2.94

1.65 0.38 0.80 _0.07 -

0.71 11.09 4.29 2.59 22.10 29.66 1.24 18.75 2.83 -

25

V

-

0.31 0.41 2.96

0.57 1.25 0.46 0.88

17.18 3.22 0.05 -

0.41 10.00 2.34 2.91 28.33 27.44 -

5

21.65 4.42 1.63 17.11 0.91 0.48 0.95

16:0 16:l 18:0 18:l 18:2 18:3 18~3 2O:l 20:2 20:3 20:3 20:4 20~4 20:5 22~4 22:5 22:5 22:6 21.55 5.8s 4.77 14.18 2.50 0.35 3.66 0.43 0.52 0.87 18.05 0.94 1.44 1.21 2.02 2.46 19.62

27.95 6.85 5.79 15.33 4.17 0.52 1.01 0.32 0.44 1.04 16.94 0.76 2.08 1.40 1.97 2.08 11.11

5

0.85 11.42 tr 2.43 0.64 6.19 4.93 25.86

1.48

1.55 -

1.75

0.96 11.36 tr* 4.65 2.26 1.18 3.40 20.18

-

9.29 1.21 14.14 13.58 5.43 0.40

2.17

17.04 3.62 7.24 17.90 4.88 0.17

25

3.23 6.79 0.33 2.51 1.06 2.28 1.06 16.54

2.82 5.88 1.03 1.64 0.73 2.58 1.29 25.14

0.74 0.94 1.44

2.17 0.50 1.50 -

14.41 1.64 12.07 19.20 4.60 -

5

17.68 5.12 11.47 16.26 5.51 -

25

Natural

Natural

-_.

.*‘j’pvpht,hG!mychthys molitrix

iw,lrna _._-.--_ glanis

5

of various fish species

0.11

0.52 5.40

0.66

0.32 1.23 1.32 1.65 22.34

-

-

0.17 1.40

1.10

34.44 3.24 7.88 16.78

25

Natural

1.13 0.41 8.31 0.96 2.33 3.50 1.46 1.39 32.38

-

25.01 1.51 6.18 12.10 1.10 0.01 0.68 -

5

IljpGplihi,“ciilmychthys no bilk

Warm-adapted fish were exposed to +5”C for 6 h before sacrifice. Only the most prominent fatty acids are listed in the table but all the fatty acids emerging from the column between 14:0 and 22:6 were involved in the calculations. tr = present in trace amounts.

7.19 2.17 19.84 0.26 0.30 1.43 6.07 0.62 8.88

24.34 3.09 10.31 18.12 2.98 0.06 0.06 1.00 0.18 5.96 1.91 16.44 0.37 0.33 0.36 4.33 0.84 7.42

Weight percentage

Fatty acid

25

25

Temperature (“C)

5

Natural

No. I

Diet

carpio

Cyprinus carpio

Cyprinus

Fish

Effect of temperature shift on liver phospholipid fatty acid compositions

TABLE VI

2

38

of fat and containing also a medium level of docosahexonoic acid in their phospholipids, rapidly accumulated this fatty acid upon cold exposure and, as Table V indicates, resembled the fish on diets IV and V in this respect within a few hours. The fish on these latter diets did not elevate the level of docosahexenoic acid in their liver total lipids (and probably in phospholipids) while exposed to cold although they continued to produce it. From Table VI it appears that other fish species respond in the same way to decrease of temperature. Thus, accumulation of docosahexenoic acid in phospholipids in response to cold appears to be the basic property of fish livers or perhaps of aquatic poikilothermic animals in general. DISCUSSION

The data presented in this paper show clearly that fish sensitively adapt their fatty acid metabolism to decrease in body temperature, and the result of these alterations in lipid metabolism is the accumulation of long chain polyunsaturated fatty acids (especially of docosahexenoic acid) in phospholipids within a short period of time. As unsaturated fatty acids are the major determinants of membrane fluidity, this response can be regarded as a manifestation of homeoviscous adaptation of membrane fluidity to the temperature first described for bacteria (Sinensky, 1974) and observed recently also in fish exposed to cold for a longer period of time (Cossins, 1977; Wodke, 1978) It has been shown for certain microorganisms that they survive even when a considerable proportion of their structural lipids undergo liquid-crystalline to solid-gel transition (McElhaney, 1974). From the fact that the fish efficiently adjusted their phospholipid fatty acid compositions to the new temperature, it can be concluded that they continuously alter the fluidity of the membrane according to the temperature. The presence of a sensitive control system of membrane fluidity in these animals is not surprising. Most cultivated fish live in relatively shallow ponds, the temperature of which many exhibit considerable diurnal variations, but also unexpected changes. Animals which are unable to adjust membrane fatty acid compositions and hence membrane fluidity to the temperature may be handicapped by such sudden changes. In fact carp maintained on diet I and unable to increase the rate of formation of polyunsaturated fatty acids in cold lost their balance and lay on the bottom of the aquaria until the water was rewarmed to 8-10°C. It has been proposed that membrane fluidity itself plays a key role in fatty acid desaturation by the unicellular eucaryotic Tetruhymena pyriformis (Kitayima and Thompson, 1977). The fatty acid composition of fish adapted to warm conditions could be manipulated by dietary means: animals maintained on high dietary intake of linolenic acid accumulated massive amounts of docosahexenoic acid in liver phospholipids. Thus formation and accumulation of this latter fatty acid, as in the rainbow trout (Caste11 et al,, 1972), appears to be the function of linolenic acid in the diet when linolenic acid is the exclusive source of 03 fatty acids. We did not carry out thermal analyses on these lipids. It is accepted, however, that the actual fluidity of phospho-

39

lipid mernbranes is dependent on the ratio of saturated to unsaturated fatty acids (Cossins, 1977). Lower ratios represent more fluid membranes. This ratio is nearly identical in carp showing no signs of essential fatty acid deficiency, and is consistently higher than in those kept on diets I and II. The same is true for rainbow trout (Caste11 et al., 1972; Yu and Sinnhuber, 1976) and carp investigated by Watanabe et al. (1975). Thus it is probable that fish sufficient in essential fatty acid maintained constant and similar membrane fluidities in spite of the vast quantities of linolenic acid taken up with the diet, whi1.e those on diets I and II possessed more fluid membranes. On the other hand, the fact that carp with high initial levels of docosahexenoic acid failed to accumulate it in liver upon exposure to cold supports the idea of involving membrane fluidity in the control of fatty acid composition in the cold, Thi;s can be also an adequate explanation of why essential fatty acid deficient carp failed to increase the production of these fatty acids when exposed to cold. Of course, the absence of appropriate precursors in the diet of the latter fish must also be considered. The observation that even the fish exhibiting the highest level of docosahexenoic acid in liver phospholipids continued to produce it when exposed to cold can be explained by assuming that the newly formed polyunsaturated fatty acid was deposited in extrahepatic tissues. Analyses on muscle and other organs will be necessary to prove this hypothesis. Evidently, the ratio of saturated to unsaturated fatty acids largely reflects the balance of the enzyme systems producing these fatty acids (i.e. fatty acid synthetase and various desaturases and chain elongases). Lower ratios can be achieved simply by reducing the activity of fatty acid synthetase. Carp on essential fatty acid deficient diets are forced to exploit this alternative: they direct less activity into palmitic acid relative to long chain polyunsaturated fatty acids than those consuming diets rich in linolenic acid. This type of membrane fluidity control is probably insufficient when the temperature decreases. Healthy fish seem to manipu,late with both enzyme systems s:imultaneously. Decrease in the rate of formation of saturated fatty acids is accompanied by increase in the rate of formation of long chain polyunsaturated fatty acids, especially of docosahexenoic acid. We believe that this fatty acid plays a special role in both membrane fluidity control and temperature adaptation processes in fish, but further investigations are necessary to clarify the biochemical and structural backgrounds of this phenomenon. As the actual level of docosahexenoic acid can be manipulated by dietary means, selection of feeds of proper fatty acid composition must have some bearing on adaptation of fish to their environment. REFERENCES Castell, J.D., Lee, D.J. and Sinnhuber, R.C., 1972. Essential fatty acids in the diet of rainbow trout (S&no gairdneri): lipid metabolism and fatty acid composition. J. Nutr., 102: 93-100. Cossins, A.IR., 1977. Adaptation of biological membranes to temperature. The effect of temperature acclimation of goldfish upon the viscosity of synaptosomal membranes. Biochim. Biophys. Acta, 470: 395-411.

40 Cossins, A.R. and Presser, C.L., 1978. Evolutionary adaptation of membranes to temperature. Proc. Natl. Acad. Sci. U.S.A., 75: 2040-2043. Farkas, T., 1971. A possible explanation for the differences in the fatty acid composition of fresh-water and marine fishes. Ann. Inst. Biol. Tihany Hung., 38: 143-152. Farkas, T., 1979. Adaptation of fatty acid composition to temperature. A study on planktonic crustaceans. Comp. Biochem. Physiol., 64B: 71-76. Farkas, T. and Csengeri, I., 1976. Biosynthesis of fatty acids by the carp, Cwrinus carPi L. in relation to environmental temperature. Lipids, 11: 401-407. Farkas, T., Csengeri, I., Majoros, F. and Olah, J., 1977. Metabolism of fatty acids in fish. I. Development of essential fatty acid deficiency in the carp, Cyprinus carpio Linnaeus 1758. Aquaculture, 11: 147-157. Farkas, T., Csengeri, I., Majoros, F. and Olah, J., 1978. Metabolism of fatty acids in fish. II. Biosynthesis of fatty acids in relation to diet in the carp, Cyprinus carpio Linnaeus 1758. Aquaculture, 14: 57-65. Folch, J., Lees, M. and Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226: 497-509. Hazel, J.Z. and Prosser, C.L., 1974. Molecular mechanism of temperature compensation in poikilotherms. Physiol. Rev., 54: 620-677. Kitayima, Y. and Thompson, G.A.. 1977. Self regulation of membrane fluidity. The effect of saturated normal and methoxy fatty acid supplementation on Tetrahymena membrane physical properties and lipid composition. Biochim. Biophys. Acta, 468: 73-80. Knipprath, W.G. and Mead, J.F., 1966a. Influence of temperature on the fatty acid pattern of mosquitofish (Gambusia affinis) and guppies (Lebistes reticulatus). Lipids, 1: 113-117. Knipprath, W.G. and Mead, J.F., 1966b. Influence of temperature on the pattern of muscle and organ lipids of rainbow trout (Salmo gairdneri). Fish Ind. Res., 3: 23-27. McElhaney, R.N., 1974. The effect of alterations in the physical state of the membrane lipids on the ability of Acheloplasma laidluwii B. to grow at various temperatures. J. Mol. Biol., 84: 145-157. Miller, N.G.A., Hill, M.W. and Smith, M.W., 1976. Positional and species analysis of membrane phospholipids extracted from goldfish adapted to different environmental temperatures. Biochim. Biophys. Acta, 455: 644-654. Patton, J.S., 1975. The effect of pressure and temperature on phospholipid and triglyceride fatty acids of fish white muscle: a comparison of deep water and surface marine fishes. Comp. Biochem. Physiol., 52B: 105-110. Reiser, R., Stevenson, B., Kayama, M., Choudhury, R.B.R. and Hood, D.W., 1963. The influence of dietary fatty acids and environmental temperature on the fatty acid composition of teleost fish. J. Am. Oil Chem. Sot., 40: 507-513. Sinensky, H., 1974. Homeovirous adaptation - a homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A., 71: 522-525. Stickney, R.R. and Andrews, J.W., 1971. Combined effects of dietary lipids and environmental temperature on growth, metabolism end body composition of channel catfish (Zctaluruspunctatus). J. Nutr., 101: 1703-1710. Watanabe, T., Takeuchi, T. and Ogino, C., 1975. Effect of methyl linoleate and linolenate on growth of carp - II. Bull. Jpn. Sot. Sci. Fish., 41: 263-269. Wodke, E., 1978. Lipid adaptation in liver mitochondrial membranes of carp acclimated to different environmental temperature. Phospholipid composition, fatty acid pattern ,and cholesterol content. Biochim. Biophys. Acta, 529: 280-291. Worthington, R.E. and Lowell, R.T., 1973. Fatty acids of channel catfish (Zctalurus punctuatus). Variance components related to diet, replications within diets, and variebility among fish. J. Fish Res. Board Can., 30: 1604-1608. Yu, T.C. and Sinnhuber, R.O., 1976. Growth response of rainbow trout (Salmo gairdneri) to dietary w3 and w6 fatty acids. Aquaculture, 8: 309-317.