Biosynthesis of Fatty Acids by the Carp, Cyprinus carpio L., in Relation to Environmental Temperature TIBOR FARKAS and ISTVAN CSENGERI,1 Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, 6701-Szeged, Hungary ABSTRACT

pounds in the right proportion in theft membranes. The phase transition point of membranes in warm blooded animals is well below the body temperature (20,21) and, except in a few cases (22), control of membrane fluidity is unnecessary. Poikilothermic animals, such as fish, do not h,~ve a constant body temperature. The adapt' tion of the physico-chemical properties of their membranes to ever-changing temperatures, therefore, has considerable survival value. This concept is supported by observations shewing that prolonged cold exposure results in an accumulation of long chain polyunsaturated fatty acids in phospholipids of fish (23-27) and other aquatic animals (28-31). Increase in activity of some membrane-bound enzymes (32,33), including desaturation of fatty acids (34), was also noted. The present paper shows that the adjustment of long chain polyunsaturated fatty acids to changes in the environmental temperature is quite rapid in fish and does not depend on the temperature history of the animals.

I n c o r p o r a t i o n in vivo of sodium 1-14C-acetate into different lipid classes and fatty acids of total lipids and phospholipids of warm adapted and cold adapted carp livers was studied at 5 C and 22 C, respectively. The fatty acid composition of total lipids and phospholipids was also determined. The level of long chain polyunsaturated fatty acids in both total lipid and phospholipid fractions was h i g h e r in cold a d a p t e d fish t h a n in w a r m a d a p t e d o n e s . T h e d i s t r i b u tion of radioactivity among different lipid classes depended only on the actual i n c o r p o r a t i o n temperature and was independent of the temperature history of the animals. Livers of fish incorporated a higher percentage of radioactivity into long chain polyunsaturated fatty acids of total lipids and phospholipids in 5 C than in 22 C. The distribution of radioactivity among different fatty acids was dependent on the experimental temperature rather than on the temperature to which the fish were adapted. The results suggest that fish are able to adjust the pattern of t h e biosynthesis of fatty acids very rapidly to the prevailing temperature and to assure by this way the proper physicochemical properties of their membranes. The possible mechanisms involved in this rapid response are discussed.

MATERIALS AND METHODS Animals

INTRODUCTION

Cholesterol content, the level of lysophospholipids, and the presence of unsaturated fatty acids in membranes are regarded as important in controIIing membrane fluidity (I-5). Increasing the amount of these compounds or the degree of unsaturation of phospholipid fatty acids results in a shift of phase transition of membranes toward lower temperature with corresponding changes in permeability, activity, and allosteric behavior of several membranebound enzymes (6-19). It is very important, therefore, for all organisms to have these com-

1permanent address: Fish Culture Research Station, 5540-Szarvas, Hungary.

T h e fish, Cyprinus carpio L., weighing 150-200g, were collected from their natural environment 2 days before the experiments. Warm adapted (WA) animals were collected in late summer (water temperature ~ 22 C) of 1974 and cold adapted animals in mid-winter (CA-I) and late winter (CA-2) of 1974-1975 (water temperature ~ 5 C). After collection, they were kept in aerated aquaria and transferred to the laboratory in nylon bags under oxygen on the day of the experiments. Sodium t - ] 4 C - a c e t a t e (sp act 1.18mCi/mmol) or sodium 2-14C-acetate (sp act 4.58 mCi/mmol) dissolved in 0.6% sodium chloride solution, was injected abdominally with the aid of a tuberculine syringe. After the injection (10~uCi/100 g body wt), the animals were placed in aquaria, set to 5 C and 22 C, respectively. They were stunned by a blow on the head 2 hr after the injection when incubated at 22 C and 6 hr after the injection when incubated at 5 C. These incubation times were established by incubating liver tissue slices at the above temperatures; the relatively long incubation at 5 C compensated

401

402

T. FARKAS AND I. CSENGERI

for the decreased metabolic activity in coId exposed animals. Three animals were used in each group. Extraction of Lipids

Livers were excised from freshly killed animals and pooled. After several rinses in ice cold physiological saline, they were blotted on filter paper and weighed. Lipid extraction was performed according to Folch et al. (35) by homogenizing the tissue in an all-glass Potter homogenizer in the presence of ice cold chlorof o r m : m e t h a n o l , 2:1. The homogenate was flushed with CO 2 and placed in a refrigerator overnight, then filtered and separated into two phases by adding 0.1 M KC1 solution. The extract was washed free of radioactivity by Folch theoretical upper phase (35) containing 0.1 M cold sodium acetate. Finally, the volume of the extract was adjusted to 5 ml with chloroform. Aliquots of this solution were taken for total lipid determination, counting, and further analyses. Separation of Lipids

Lipid class separation was performed by thin layer chromatography (TLC) on Silica Gel G plates using hexane:ethyl ether:acetic acid, 85:15:1, as solvent. In some cases, the separat i o n s were performed using the two-step developing techniques employed by Salle et al. (36). Several thousands of counts were applied to the plate by a micrometer syringe. The chromatograms were visualized by iodine vapor or by spraying with 0.1% Rhodamine B in ethanol and detecting under ultraviolet light. When the phospholipids were subjected to gas chromatography, no staining was employed at all; the origin was scraped directly into methylation amp oules. Gas Liquid Chromatography IGLC)

Methyl esters were prepared by transesterification in absolute methanol containing 5% hydrochloric acid, in ampoules sealed under CO2, at 80 C. The analyses were carried out on a JGC 1100 gas chromatograph equipped with flame ionization detector. The 6 ft long stainless steel column, 0.3 cm inside diameter, was packed with 15% diethylene glycol succinate on Gas Chrom P (Applied Science Lab., State College, PA), 100-200 mesh. Tile column temperature was 180 C when analytical runs were performed. For determination of radioactivity of fatty acids, a coiled glass column (0.6 cm inside diameter) was employed. The column was split before its outlet in such a way that only 25% of the eluate escaped to the detector while 75% was trapped on cotton LIPIDS, VOL. 11, NO. 5

woo1. Preparative runs were programmed from 140 to 180 C at a rate of 1 C/min to ensure the complete separation of palmitoleic and oleic acids from palmitic and stearic acids, respectively. The samples were run in two or three replicates. Peaks were identified by using suitable standards (Applied Science Lab. N ~ K-108), by secondary standards (cod liver oil), and by plotting the logarithm of the relative retention times versus the number of carbon atoms in the molecule. Quantitation was performed by the triangulation techniques. The accuracy of the determination was > 5% in the case of major fatty acids and ca. 20% in the case of minor ones. Determination of Radioactivity

Known aliquots of the chloroform extract were pipetted directly into counting vials and, after evaporation of the solvent, were dissolved in scintillation cocktail. Spots from the thin layer plates were scraped directly into counting vials. When the plates were visualized by iodine vapors, the iodine was allowed to evaporate from the spots before counting. The cotton on which the eluates were trapped, when the separation was performed by GLC, was placed w i t h o u t prior elution into counting vials. Toluene scintillation cocktail (4% PPO and 0.2% POPOP) was used throughout the experiments. Counting was performed by an Isocap 300 Nuclear Chicago or a Tri Carb Liquid S c i n t i l l a t i o n S p e c t r o m e t e r . Counts were corrected for quenching using sample channel ratio techniques and for counting efficiency. R ESU LTS Fatty Acid Composition of Warm and Cold Adapted Fish

Total fatty acids extracted from livers of cold adapted animals contained more a r a c h i d o n i c acid ( 2 0 : 4 6 0 6 ) a n d d o c o s a hexaenoic acid (22:6603) than those obtained from warm adapted animals (Table I). The increase in the level of arachidonic acid was ca. ten-fold and, in the level of docosahexaenoic acid, ca. two-fold in CA-2 animals, as compared with WA fish. During cold adaptation, the level of palmitic acid (16:0) was reduced by ca. 50%. In contrast, there was an increase in the content of stearic acid. Oleic acid (18:1) did not exhibit any change during this period. Phospholipids exhibited changes similar to the total fatty acids. However, they were richer in arachidonic and docosahexaenoic acids. These resu!ts are comparable to those obtained f o r Garnbusia affinis, Lebistes reticulatus, Salmo gairdneri, and Carassius aureatus (23,27),

TEMPERATURE AND FA BIOSYNTHESIS IN THE CARP

403

TABLE I Fatty Acid Composition (% by wt) of Liver Total Fatty Acids and Phospholipids of Cold and Warm Adapted Carp a Total fatty acids Fatty acids

WA

14:0

CA-I

1.7

0.8

Phospholipids CA-2

WA

0.8

0.5 32.0 6.4 5.4 10.4 3.1 0.7 2.7 1.1 tr 0.5 16.8 tr tr 3.1 3.9 10.3

1 6:0

20.3

32.2

11.6

16:1 e67 18:0 18:16o9 18:2666 18:36o6 18 : 3o93 20:2666 20:3666 20:3663 20:4666 20:4663 20:5663 22:40)6 22:5666 22:6663

10.4 6.7 35.7 11.3 0.7 4.5 0.6 0.4 0.5 1.1 0.1 0.1 0.2 0,6 2,8

6.6 6.3 22.0 9.5 0.6 4.2 0.1 0.2 0.9 8.9 tr 0.5 0.7 0.8 4.6

3.8 9.8 34.0 10.9 0.4 3.6 0.t 0.2 0.5 10.8 tr 1.2 1.2 3.8 6.0

CA-1

CA-2

0.7

0.5

18.1

14.6

4.7 7.3 10.5 4.0 1.0 2.7 1.2 1.3 2.3 2 3.0 0.3 0.3 2.4 2.9 1 3.7

4.7 9.1 15.7 0.5 0~2 1,4 0.3 0.3 0.6 25.4 0.1 0.1 1.2 2.1 19.7

aWA = warm adapted fish; CA-l, CA-2 = cold adapted fish. WA animals were collected in late summer of 1974, CA-1 and CA-2 in mid and late winter of 1974-1975, respectively. TABLE II Incorporation of Radioacetate into Liver Lipids of Carp at Different Temperatures Animals a Temperature (C) nMol of acetate incorporated by 100 mg of liver

WA 22

5

10.3

4.2

4.3 2.5 28.0 0.8 2.2 62.2

17.3 1.8 29.6 1.9 4.4 44.9

CA-1 5

CA-2 5

CA-1 22

CA-2 22

CA-2 b 22

4.6

5.2

15.7

4.8

8.9

1.6 15.2 5.8 1 3.7 1.2 62.5

1.2 14.0 3.8 9.1 0.5 71.2

Percent distribution of radioactivity Cholesterol esters Triglycerides Free fatty acids Cholesterol Diglyeerides Phospholipids

15.0 35.0 0.6 1.3 5.9 41.3

1.2 24.5 6.1 33.8 2.1 32.0

5.0 30.0 0.2 0.6 1.9 62,1

aWA = warm adapted fish; CA-l, CA-2 = cold adapted fish. WA and CA-1 animals received 1-14C-sodium acetate; CA-2 animals 2-14C-sodium acetate 2 hr (experiments at 22 C) or 6 hr (experiments at 5 C) before sacrifice. bAnimals received glucose before the injection of acetate. with the exception that the changes evoked by t h e d e c r e a s e in e n v i r o n m e n t a l t e m p e r a t u r e w e r e more dramatic than with the above animals.

Incorporation of 1-14C-Acetate into Liver Lipids A f t e r i n j e c t i o n o f s o d i u m 1 -I 4 C - a c e t a t e , t h e fiver lipids b e c a m e h i g h l y l a b e l e d . Livers o f W A fish i n c o r p o r a t e d ca. 60% less label at 5 C t h a n at +22 C (4.2 nmol/100mg liver v e r s u s 10.3 n m o l ) . T h e r a t e o f b i o s y n t h e s i s o f lipids b y CA-1 a n d CA-2 a n i m a l s at +5 C w a s c o m p a r a b l e t o t h a t o b t a i n e d f o r W A fish at +5 C ( T a b l e II). I n c u b a t i o n o f CA-1 a n i m a l s w i t h a c e t a t e at 22 C r e s u l t e d in a s u b s t a n t i a l i n c r e a s e in t h e

i n c o r p o r a t i o n o f a c e t a t e . CA-2 a n i m a l s failed t o synthesize m o r e lipid w h e n t r e a t e d w i t h 2-I4C-acetate at 2 2 C unless glucose was injected abdominally before the experiment ( T a b l e II). CA-1 a n d CA-2 a n i m a l s h a d a l o w e r p r o p o r t i o n o f r a d i o a c t i v i t y in t h e i r p h o s p h o f i p i d s t h a n did t h e i r WA c o u n t e r p a r t s a n d m o r e a c t i v i t y in c h o l e s t e r o l . S i m i l a r l y , WA f i s h h a d a l o w e r p r o p o r t i o n o f r a d i o a c t i v i t y in p h o s p h o l i p i d s w h e n i n j e c t e d w i t h a c e t a t e in t h e cold. T h e p r o p o r t i o n o f r a d i o a c t i v i t y in p h o s p h o l i p i d s a f t e r i n j e c t i o n o f CA a n i m a l s w i t h a c e t a t e in t h e w a r m w a s v e r y s i m i l a r t o t h e v a l u e s obt a i n e d w i t h W A f i s h at 22 C. T h e f r e e f a t t y acid LIPIDS, VOL. 11, NO. 5

T. FARKAS AND I. CSENGERI

404

TABLE III Distribution o f Radioactivity a m o n g T o t a l Fatty A c i d s o f Livers o f Warm and Cold A d a p t e d Carp at Different T e m p e r a t u r e s

Animals a Temperature (C) nMol o f acetate i n c o r p o r a t e d by 100 mg o f liver c

WA 22

5

16:0 16:1 o97 18:0 18:lo99 18:2o96 18:3o96 18:3o93 20:2 + 20:3 20:4o96 20:4o93 20:5o93 22:4t~6 22:5(o6 22:5o93 22 : 6o93

CA-2 5

CA-I 22

CA-2 22

CA-2 b 22

3.9

3.4

14.9

4.1

8.0

1.7 35.9 4.4 28.8 2.6 2.1 2.2 3.4 6.1 3.0 2.0 1.6 ND 2.2 2.3 1.7

9.8

3.4

3.9

6.3

5.7

4.2

4.6

4.8

49.6 3.9 13.4 7.9 0.9 1.6 5.6 3.6 2.3 ND 0.9 2.1 1.0 1.1 2.0

15.2 1.8 14.3 11.1 1.3 5.9 14.8 8.3 5.4 ND 1.3

14.7 3.0 15.2 4.8 2.5 2.1 12.7 13.1 5.9 4.2 2.9

11.4 2.3 7.6 5.4 2.8 2.1 6.0 12.6 7.2 3.1 5.2

45.2 4.6 7.0 9.2 2.9 1.4 5.4 6.4 2.5 1.9 0.9

5.2

5.4

5.4

1.8

2.4 4.3 2.2

1.9 4.3 3.6

3.8 10.0 10.6

2.0 1.9 2.0

32.0 3.1 13.4 4.1 2.8 2.7 5.7 13.4 3.8 2.3 3.1 ND 2.9 3.3 2.2

Fatty acids