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Brain lipid fatty acids and temperature acclimation
Comp. Biochem. Physiol., 1964, Vol. 11, pp. 303 to 309. Pergamon Press Ltd. Printed in Great Britain
BRAIN LIPID FATTY ACIDS AND TEMPERATURE ACCLIMATION* P A T R I C I A V. J O H N S T O N
and B E T T Y I. R O O T S
Department of Anatomy, University College London, Gower Street, London, W.C.1 (Received 1 November 1963)
A b s t r a c t - - 1 . Goldfish (Carassius auratus L.) were acclimated to temperatures of 5, 15, 25 and 30°C, and the total amount and the fatty acid composition of their brain lipids were determined. 2. The total amount of lipid increased with decreasing acclimation temperature. 3. There was an overall tendency for the degree of unsaturation of the fatty acids to increase when the acclimation temperature was decreased. 4. The greatest differences were observed in the relative amounts of stearic, arachidonic and docosapenta (or hexa) enoic acids. 5. Less pronounced changes occurred in the relative amounts of palmitoleic, linoleic and linolenic acids. 6. It is suggested that acclimation involves the ability to control the degree of unsaturation of cellular lipids in order to maintain a specific liquid-crystalline state of cellular "membranes". INTRODUCTION
A NUMBERof workers have established the existence of a relationship between the degree of saturation of b o d y fatty acids and the ability of animals to acclimate to different environmental temperatures. H o a r & Cottle (1952) have shown that the lipid content of goldfish (Carassius auratus L.) decreases with a rise in environmental t e m p e r a t u r e and that the degree of unsaturation of the lipid increases at lower temperatures. Fraenkel & H o p f (1940) studied the adaptability to high t e m p e r a t u r e and the nature of the phosphatides in blowfly larvae (Calliphora erythrocephala Meigen and Phormia terra-novae R.D.), and found that the degree of unsaturation of the phosphatides was dependent on the breeding temperature. House et al. (1958) demonstrated that the resistance of larvae of Pseudosarcophaga affinis Fall. to high temperature was increased by thermal conditioning and by dietary lipids, the highest resistance being found when the larvae were reared on a diet rich in saturated fatty acids. By studies on the effect of low environmental t e m p e r a t u r e on depot fat in relation to hibernation Fawcett & L y m a n (1954) showed that in hamsters low environmental temperatures led to a decrease in the saturation of depot fat, but in the rat no significant changes occurred. * This work was carried out while the authors held appointments at the University of Illinois, Urbana, Illinois, U.S.A. 303
304
PATRICIA V. JOHNSTON AND BETTY I. ROOTS
It has previously been reported (Roots & Prosser, 1962) that temperature acclimation in fish involves modifications of the nervous system which permit normal functioning at higher or lower temperatures than before acclimation. Whether or not these modifications are accompanied by changes in the degree of saturation of brain lipid fatty acids, therefore, became of interest. The results of such a study are reported here and their significance is discussed.
MATERIALS AND METHODS
Source, maintenance and acclimation offish Goldfish (Carassius auratus L.), 11-2 years old and 4-5 in. in total length (No. 1 Pool fish), were obtained from the Auburndale Company, Chicago. The fish were kept under conditions of constant length of day and acclimated to temperatures of 5, 15, 25 and 30°C as described previously (Roots & Prosser, 1962). Fish were fed ad libitum with Glencoe Mills Company ~- in. pellet trout food.
Extraction of lipid from brains The brains were excised immediately after decapitation and were weighed rapidly. They were then kept frozen in glass vials in solid carbon dioxide until the lipid could be extracted 2-3 hr later. The lipid was extracted at room temperature with 4 x 10 ml of chloroform-methanol 2:1 (v/v). The combined extracts were washed with 0.2 vol water (Folch et al., 1957) and the solvent was removed under vacuum on a rotary evaporator. All manipulations of the lipid were carried out under an atmosphere of oxygen-free nitrogen.
Gas-liquid chromatography (GLC) The methyl esters of the brain lipid fatty acids were obtained by direct methanolysis as previously described by Johnston et al. (1961). Gas chromatographic analysis of the methyl esters was carried out using a model 90C Aerograph (Wilkens Instrument & Research, Calif.) with thermal conductivity cells as detectors. The column employed was a 10 ft, 20% diethylene glycol succinate (DEGS) with a ]: in. O.U. All analyses were carried out at temperatures between 190-215°C. Identification of peaks was made by comparison of retention times with known standards, by use of internal standards and, in cases where no standards were available, by use of the "carbon number" (Woodford & van Gent, 1960). Quantitative determinations were made on the basis of peak area measured with a planimeter. Under the conditions used for GLC the methyl esters of non-hydroxylated fatty acids only were eluted and no attempt was made to analyse the a hydroxy fatty acids which would occur predominantly in the cerebrosides (Kishimoto & Radin, 1959) and would only be eluted from a DEGS column in the form of methoxy derivatives of their methyl esters (Johnston & Kummerow, 1960). Future references to fatty acids, therefore, refer to the nonhydroxylated acids only. Statistical analysis of the results was carried out according to Snedecor (1956).
305
B R A I N L I P I D FATTY ACIDS AND TEMPERATURE A C C L I M A T I O N
RESULTS Temperature acclimation in goldfish had a decided effect on both the total amount of brain lipid and its fatty acid composition. T h e amount of lipid in the brain increased with a decrease in the acclimation temperature. Goldfish acclimated to 5, 15, 25 and 30°C had 9.32+1.31, 8.98+0.82, 8.59+1-38 and 8.30 + 0.54 per cent lipid in the brain respectively. TABLE
1--GAS-LIQUID
C H R O M A T O G R A P H Y OF THE M E T H Y L ESTERS OF N O N - H Y D R O X Y L A T E D
FATTY ACIDS OF B R A I N L I P I D FROM F I S H A C C L I M A T E D TO D I F F E R E N T TEMPERATURES
% Fatty acid (peak area)
Fatty acid 5 °* 12-16 16 : 0t 16 : 1 18 : 0 18 : 1 18 : 2 18 : 3, 20:0 20 : 4 22 : 5 or 6§ Othersll
6-16 24-44 _+0.49++ 9-76 ± 0.64 13.01 ± 2.42 25.55 ± 3"41 1.61 ± 0.74 0.93 4-13 ± 0.25 2.45 ± 0"19 10"48
15°
25 °
30°
6.41 24-36 + 0.46 8-84 ± 0.46 14.41 ± 0-56 21.48 _+0.41 2.68 ± 1.86 0.91 4.25 ± 1-12 1"99 ± 0-42 14-51
5"73 25"49 + 0-26 8"48 ± 0-57 16.61 ± 0.47 22-94 ± 0.62 2.05 ± 0-22 0.62 2.51 ± 1.06 1"76 ± 1"45 13"91
5.93 24.78 + 0-84 7.83 +_1-84 18-33 ± 1.0 24.51 ± 1"29 0.87 ± 0.15 0.77 1.29 ± 0.42 0"47 ± 0'43 13"26
* Acclimation temperature °C. "~Figure preceding colon indicates chain length, figure after colon the number of double bonds. ++Standard deviation of the mean (3-5 samples). § No standard fatty acid available, whether 5 or 6 double bonds uncertain. 1] Minor fatty acids including branched chain and unidentified acids of chain length beyond 22.
T h e effect of acclimation on the fatty acid composition is summarized in Table 1. T h e overall tendency was for a greater degree of unsaturation at lower acclimation temperatures. T h e greatest differences were observed in the relative amounts of stearic (18:0), arachidonic (20:4) and docosapenta (or hexa) enoic (22 : 5 or 6) acids. T h e differences in stearic acid content between the brain lipid of the 5 and 30°C, 15 and 30°C, and the 15 and 25°C fish were all highly significant, while differences between the 25 and 30°C, and the 5 and 25°C fish were less significant (Table 2). Arachidonic acid was significantly higher in the brain lipid of 5 and 15°C as compared to 30°C fish (Table 2). T h e amount of docosapenta (hexa) enoic acid increased with a decrease in the acclimation temperature, the difference between the 5 and 30°C fish brain lipid being highly significant (Table 2). Less pronounced differences were observed in the relative amounts of palmitoleic (16 : 1), linoleic (18 : 2) and linolenic (18 : 3) acids. T h e palmitoleic acid content
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PATRICIA V . J O H N S T O N AND BETTY I. R O O T S
showed a distinct tendency to increase with a decrease in the acclimation temperature (Table 1). T h e amounts of oleic (18 : 1) and linoleic (18:2) did not, as may have been expected, progressively increase with the decrease in acclimation temperature. T h e oleic acid content of brain lipid from 15 and 25°C fish tended to be lower and the linoleic acid content higher than brain lipid from fish acclimated to either of the extreme temperatures (Table 1). TABLE 2--TESTS
FOR SIGNIFICANCE OF DIFFERENCES IN BRAIN LIPID FATTY ACID COMPOSITION ASSOCIATED W I T H ACCLIMATION (SNEDECOR, 1956)
Fatty acid Palmitoleic (16 : 1) Stearic (18:0)
Arachidonic (20:4)
Docosapenta (hexa) enoic 22 : 5 or 6
Groups being compared 5° and 5° and 15 ~ and 15 ° and 5'~ and 25 Qand 5' and 15 ° and 5" and 15 ~ and
30 ° 30 ° 30° 25 ° 25 ° 30 ~ 25 ~ 25 ~ 30 * 30 °
5:' and 30 °
"t" 1-68 3.45 4"90 3-55 2'56 2-72 2"62 1-98 10"10 4.27 4"69
p >0.10 0.025-0"05 0"01 0.025 0"05-0"10 0.05-0.10 0.05-0.10 >0.10 0'001 0.025 0"01
In addition to the brain lipid analyses a note was also made of the relative amount of lipid in the livers of the same fish. It was observed that in contrast to the results for brain, the lipid content of the liver tended to decrease with a decrease in the acclimation temperature. T h e lipid content of the livers of fish acclimated to 5, 15, 25 and 30°C were 1.76, 2.76, 3.04 and 3"69 per cent respectively. DISCUSSION T h e preceding results are in general agreement with previous work that acclimation of animals to low temperatures leads to an increase in the degree of unsaturation of carcass fat. However, the present study is more comprehensive in that the use of G L C rather than the non-specific iodine number has made it clear that the changes, at least in the brain, involve predominantly the polyunsaturated fatty acids of twenty carbons or more. Furthermore, the observation that the amount of lipid in the brain was higher at lower acclimation temperatures, whereas the opposite trend was shown in the liver, is a clear indication that in order to elucidate the mechanisms involved in the complex process of acclimation, the roles of individual systems must be studied. I f a relationship between changes in lipid fatty acid composition and temperature acclimation exists, it should be possible to assign to lipids a definite role in the process. Since, in contrast to previous work, this study was confined to the lipids
BRAIN LIPID FATTY ACIDS AND TEMPERATURE ACCLIMATION
307
of the central nervous system, in which it had previously been shown that modifications occurred under identical acclimation conditions (Roots & Prosser, 1962), it is possible to make some suggestions regarding the functions of lipids in the nervous system and the necessity for their alteration in the acclimated animal. The following hypothesis requires two assumptions for its subsequent development. First, the surface of the neuron is considered in terms of a lyotropic mesomorphic or liquid-crystalline system. Furthermore, the ion movements taking place in nervous activity are considered as secondary to electronic conduction and developed potentials due, not to ionic diffusion alone, but to redox potentials and semi-conductivity phenomena. The surface of the neuron is considered to be a lipoprotein liquid-crystalline system dependent for its integrity on the composition of the intra- and extracellular milieux. The physical properties of this system will, to a considerable extent, be a function of those of the fatty acid residues of the constituent phospholipids. These properties will be determined by the geometry, degree of unsaturation, double bond position, degree of chain branching and presence of hydroxyl groups in the fatty acids as well as by the isomeric and polymorphic forms of the phospholipids. A resting potential across such a system may be considered as an asymmetric barrier potential developed by selective ion binding such that an electron excess develops on the inside. Any event which altered conditions on either side would lead to a shift in the membrane potential, The arrival of quanta of acetylcholine from the presynaptic terminal provides one such event. Molecules of acetylcholine by accepting electrons, probably by "charge transfer" from the postsynaptic structure, would cause a shift in the membrane potential. The resultant change in the electric field influencing the "membrane" may trigger the following sequence of events: 1. The release of ions from their binding sites. 2. Conformational changes in the constituents of the lipoprotein system leading to a phase transition. From observations by Luzzati & Husson (1962) and Stoeckenius (1962) on liquid-crystalline states of lipid-water systems it may be deduced that such a transition may be to the crystalline-like coagel state. 3. Increased permeability of the matrix to ions such as Na +. If the local disturbances at the postsynaptic membrane reach a threshold value, the current may be propagated in two ways, either by electronic resonance through molecules containing suitable double bond systems or electronic conduction through the lipoprotein matrix. Once effectively triggered, the electron flow would lead to further increased ion mobility followed by new binding states of the ions on both sides, depolarization and eventually complete reversal of membrane polarity. The system may be visualized as being coupled to ATP hydrolysis brought about as a consequence of electron and ion movements across the "membrane". Mitchell (1961) has recently shown in detail how such coupling between transport and metabolism may occur. The effective functioning of the above system is dependent on the maintenance of the liquid-crystalline state of the resting membrane which is, in turn, dependent on the temperature and on the concentration and nature of the lipid residues of the
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PATRICIA V. JOHNSTON AND BETTY I. ROOTS
lipoprotein. A particular fatty acid composition and distribution at a specific temperature may thus provide an appropriate solid-liquid ratio, degree of expansibility and hydrophilic properties necessary for the maintenance of a liquidcrystalline system which will undergo a suitable phase transition when influenced by an electrical event. However, a decrease in the environmental temperature of the same system would cause a decrease in thermal agitation and a consequent increase in the lateral attractions of molecules producing either an undesirable phase transition or an inability to undergo a particular transition. Since double bonds attract more water than do saturated linkages, and the maintenance of a liquid-crystalline state is highly dependent on the degree of hydration at a particular temperature, an increase in the degree of unsaturation of the lipids presents a means for maintaining the system near the critical point of a phase transition. An increase in the concentration of lipid may also be necessary for the maintenance of this particular state. If the environmental temperature of the same system was increased, the consequent increase in thermal agitation might lead to a breakdown of the lateral attractions between molecules and disruption of the phase. In this case additional cohesiveness may be achieved by a relative decrease in the number of unsaturated linkages, the consequent water loss leading to a less expanded state of the phase. It follows that if the environmental temperature of the proposed system is changed, adjustments in the structure of the lipoprotein must also facilitate the maintenance of the necessary features for electronic conduction, such as hydrogen bonding and suitable systems for electronic resonance. If a poikilotherm is to accommodate either a lowering or raising of its environmental temperature, it must possess the ability to control the degree of unsaturation of its cellular lipids. The mechanism by which this is achieved is obscure and may depend on a temperature-sensitive reaction in fatty acid synthesis. Such a reaction has recently been suggested as the primary cause of the effect of temperature on the fatty acid composition of certain micro-organisms (Bishop & Still, 1963). Since the cerebrosides and other myelin lipids do contain considerable quantities of non-hydroxylated fatty acids, the results obtained in this study may reflect changes occurring in myelin as well as in the lipids of neuronal and glial elements. However, the theories postulated were confined to changes in the neuronal lipids since it seems reasonable to assume that appropriate structural adjustments to environmental change are made in the myelin lipids which are, in effect, derivatives of the Schwann cells and glial elements. The complete elucidation of the mechanisms involved in acclimation may, however, only be obtained by detailed studies of the molecular architecture of the specific cells and subcellular elements involved. REFERENCES BISHOP D. G. • STILL J. L. (1963) Fatty acid metabolism in Serratia marcescens. IV. T h e effect of temperature on fatty acid composition. J. Lipid Res. 4, 87-90. F.*WeETT D. W. & LYMAN C. P. (1954) T h e effect of low environmental temperature of depot fat in relation to hibernation. J. Physiol. 126, 235-237.
BRAIN L I P I D F A T T Y ACIDS A N D TEMPERATURE A C C L I M A T I O N
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FOLCH J . , LEES M. • SLOANE STANLEY G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. 07. Biol. Chem. 226, 497-509. FRAENKELG. & HOPF H. S. (1940) T h e physiological action of abnormally high temperatures on poikilothermic animals. I. Temperature adaptation and the degree of saturation of the phosphatides. Biochem.07. 34, 1085-1092. HOAR W. S. & COTTLE M. K. (1952) Some effects of temperature acclimatization on the chemical constitution of goldfish tissues. Canad. 07. Zool. 30, 49-54. HOUSE H. L., RIORDAN D. F. & BARLOWJ. S. (1958) Effects of thermal conditioning and of degree of saturation of dietary lipids on resistance of an insect to high temperature. Canad. 07. Zool. 36, 629-632. JOHNSTON P. V., KOPACZYKK. C. & KUMMEROWF. A. (1961) Effect of pyridoxine deficiency on fatty acid composition of carcass and brain lipids in the rat. 07. Nutr. 74, 96-102. JOHNSTON P. V. & KUMMEROWF. A. (1960) Gas-liquid chromatography of methyl esters of fatty acids from human and chicken brain lipids. Proc. Soc. Exp. Biol. Med. 104, 201-205. KISHIMOTOY. & RADIN N. S. (1959) Isolation and determination methods for brain cerebrosides, hydroxy fatty acids and unsaturated and saturated fatty acids. 07. Lipid Res. I, 72-78. LUZZATI V. & HUSSON F. (1962) The structure of liquid-crystalline phases of lipid-water systems. 07. Cell. Biol. 12, 207-219. MITCHELL P. (1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature, Lond. 191, 144-148. RooTs B. I. & PROSSER C. L. (1962) Temperature acclimation and the nervous system in fish. 07. exp. Biol. 39, 617-629. SNEDECOR G. W. (1956) Statistical Methods, 5th ed. Iowa State College, Ames, Iowa. STOECKENIUS W. (1962) Some electron microscopical observations on liquid-crystalline phases in lipid-water systems. 07. Cell. Biol. 12, 221-229. WOODFORD F. P. & VAN GENT C. M. (1960) Gas-liquid chromatography of fatty acid methyl esters: T h e "carbon n u m b e r " as a parameter for comparison of columns. J. Lipid Res. 1, 188-189.
BRAIN LIPID FATTY ACIDS AND TEMPERATURE ACCLIMATION* P A T R I C I A V. J O H N S T O N
and B E T T Y I. R O O T S
Department of Anatomy, University College London, Gower Street, London, W.C.1 (Received 1 November 1963)
A b s t r a c t - - 1 . Goldfish (Carassius auratus L.) were acclimated to temperatures of 5, 15, 25 and 30°C, and the total amount and the fatty acid composition of their brain lipids were determined. 2. The total amount of lipid increased with decreasing acclimation temperature. 3. There was an overall tendency for the degree of unsaturation of the fatty acids to increase when the acclimation temperature was decreased. 4. The greatest differences were observed in the relative amounts of stearic, arachidonic and docosapenta (or hexa) enoic acids. 5. Less pronounced changes occurred in the relative amounts of palmitoleic, linoleic and linolenic acids. 6. It is suggested that acclimation involves the ability to control the degree of unsaturation of cellular lipids in order to maintain a specific liquid-crystalline state of cellular "membranes". INTRODUCTION
A NUMBERof workers have established the existence of a relationship between the degree of saturation of b o d y fatty acids and the ability of animals to acclimate to different environmental temperatures. H o a r & Cottle (1952) have shown that the lipid content of goldfish (Carassius auratus L.) decreases with a rise in environmental t e m p e r a t u r e and that the degree of unsaturation of the lipid increases at lower temperatures. Fraenkel & H o p f (1940) studied the adaptability to high t e m p e r a t u r e and the nature of the phosphatides in blowfly larvae (Calliphora erythrocephala Meigen and Phormia terra-novae R.D.), and found that the degree of unsaturation of the phosphatides was dependent on the breeding temperature. House et al. (1958) demonstrated that the resistance of larvae of Pseudosarcophaga affinis Fall. to high temperature was increased by thermal conditioning and by dietary lipids, the highest resistance being found when the larvae were reared on a diet rich in saturated fatty acids. By studies on the effect of low environmental t e m p e r a t u r e on depot fat in relation to hibernation Fawcett & L y m a n (1954) showed that in hamsters low environmental temperatures led to a decrease in the saturation of depot fat, but in the rat no significant changes occurred. * This work was carried out while the authors held appointments at the University of Illinois, Urbana, Illinois, U.S.A. 303
304
PATRICIA V. JOHNSTON AND BETTY I. ROOTS
It has previously been reported (Roots & Prosser, 1962) that temperature acclimation in fish involves modifications of the nervous system which permit normal functioning at higher or lower temperatures than before acclimation. Whether or not these modifications are accompanied by changes in the degree of saturation of brain lipid fatty acids, therefore, became of interest. The results of such a study are reported here and their significance is discussed.
MATERIALS AND METHODS
Source, maintenance and acclimation offish Goldfish (Carassius auratus L.), 11-2 years old and 4-5 in. in total length (No. 1 Pool fish), were obtained from the Auburndale Company, Chicago. The fish were kept under conditions of constant length of day and acclimated to temperatures of 5, 15, 25 and 30°C as described previously (Roots & Prosser, 1962). Fish were fed ad libitum with Glencoe Mills Company ~- in. pellet trout food.
Extraction of lipid from brains The brains were excised immediately after decapitation and were weighed rapidly. They were then kept frozen in glass vials in solid carbon dioxide until the lipid could be extracted 2-3 hr later. The lipid was extracted at room temperature with 4 x 10 ml of chloroform-methanol 2:1 (v/v). The combined extracts were washed with 0.2 vol water (Folch et al., 1957) and the solvent was removed under vacuum on a rotary evaporator. All manipulations of the lipid were carried out under an atmosphere of oxygen-free nitrogen.
Gas-liquid chromatography (GLC) The methyl esters of the brain lipid fatty acids were obtained by direct methanolysis as previously described by Johnston et al. (1961). Gas chromatographic analysis of the methyl esters was carried out using a model 90C Aerograph (Wilkens Instrument & Research, Calif.) with thermal conductivity cells as detectors. The column employed was a 10 ft, 20% diethylene glycol succinate (DEGS) with a ]: in. O.U. All analyses were carried out at temperatures between 190-215°C. Identification of peaks was made by comparison of retention times with known standards, by use of internal standards and, in cases where no standards were available, by use of the "carbon number" (Woodford & van Gent, 1960). Quantitative determinations were made on the basis of peak area measured with a planimeter. Under the conditions used for GLC the methyl esters of non-hydroxylated fatty acids only were eluted and no attempt was made to analyse the a hydroxy fatty acids which would occur predominantly in the cerebrosides (Kishimoto & Radin, 1959) and would only be eluted from a DEGS column in the form of methoxy derivatives of their methyl esters (Johnston & Kummerow, 1960). Future references to fatty acids, therefore, refer to the nonhydroxylated acids only. Statistical analysis of the results was carried out according to Snedecor (1956).
305
B R A I N L I P I D FATTY ACIDS AND TEMPERATURE A C C L I M A T I O N
RESULTS Temperature acclimation in goldfish had a decided effect on both the total amount of brain lipid and its fatty acid composition. T h e amount of lipid in the brain increased with a decrease in the acclimation temperature. Goldfish acclimated to 5, 15, 25 and 30°C had 9.32+1.31, 8.98+0.82, 8.59+1-38 and 8.30 + 0.54 per cent lipid in the brain respectively. TABLE
1--GAS-LIQUID
C H R O M A T O G R A P H Y OF THE M E T H Y L ESTERS OF N O N - H Y D R O X Y L A T E D
FATTY ACIDS OF B R A I N L I P I D FROM F I S H A C C L I M A T E D TO D I F F E R E N T TEMPERATURES
% Fatty acid (peak area)
Fatty acid 5 °* 12-16 16 : 0t 16 : 1 18 : 0 18 : 1 18 : 2 18 : 3, 20:0 20 : 4 22 : 5 or 6§ Othersll
6-16 24-44 _+0.49++ 9-76 ± 0.64 13.01 ± 2.42 25.55 ± 3"41 1.61 ± 0.74 0.93 4-13 ± 0.25 2.45 ± 0"19 10"48
15°
25 °
30°
6.41 24-36 + 0.46 8-84 ± 0.46 14.41 ± 0-56 21.48 _+0.41 2.68 ± 1.86 0.91 4.25 ± 1-12 1"99 ± 0-42 14-51
5"73 25"49 + 0-26 8"48 ± 0-57 16.61 ± 0.47 22-94 ± 0.62 2.05 ± 0-22 0.62 2.51 ± 1.06 1"76 ± 1"45 13"91
5.93 24.78 + 0-84 7.83 +_1-84 18-33 ± 1.0 24.51 ± 1"29 0.87 ± 0.15 0.77 1.29 ± 0.42 0"47 ± 0'43 13"26
* Acclimation temperature °C. "~Figure preceding colon indicates chain length, figure after colon the number of double bonds. ++Standard deviation of the mean (3-5 samples). § No standard fatty acid available, whether 5 or 6 double bonds uncertain. 1] Minor fatty acids including branched chain and unidentified acids of chain length beyond 22.
T h e effect of acclimation on the fatty acid composition is summarized in Table 1. T h e overall tendency was for a greater degree of unsaturation at lower acclimation temperatures. T h e greatest differences were observed in the relative amounts of stearic (18:0), arachidonic (20:4) and docosapenta (or hexa) enoic (22 : 5 or 6) acids. T h e differences in stearic acid content between the brain lipid of the 5 and 30°C, 15 and 30°C, and the 15 and 25°C fish were all highly significant, while differences between the 25 and 30°C, and the 5 and 25°C fish were less significant (Table 2). Arachidonic acid was significantly higher in the brain lipid of 5 and 15°C as compared to 30°C fish (Table 2). T h e amount of docosapenta (hexa) enoic acid increased with a decrease in the acclimation temperature, the difference between the 5 and 30°C fish brain lipid being highly significant (Table 2). Less pronounced differences were observed in the relative amounts of palmitoleic (16 : 1), linoleic (18 : 2) and linolenic (18 : 3) acids. T h e palmitoleic acid content
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PATRICIA V . J O H N S T O N AND BETTY I. R O O T S
showed a distinct tendency to increase with a decrease in the acclimation temperature (Table 1). T h e amounts of oleic (18 : 1) and linoleic (18:2) did not, as may have been expected, progressively increase with the decrease in acclimation temperature. T h e oleic acid content of brain lipid from 15 and 25°C fish tended to be lower and the linoleic acid content higher than brain lipid from fish acclimated to either of the extreme temperatures (Table 1). TABLE 2--TESTS
FOR SIGNIFICANCE OF DIFFERENCES IN BRAIN LIPID FATTY ACID COMPOSITION ASSOCIATED W I T H ACCLIMATION (SNEDECOR, 1956)
Fatty acid Palmitoleic (16 : 1) Stearic (18:0)
Arachidonic (20:4)
Docosapenta (hexa) enoic 22 : 5 or 6
Groups being compared 5° and 5° and 15 ~ and 15 ° and 5'~ and 25 Qand 5' and 15 ° and 5" and 15 ~ and
30 ° 30 ° 30° 25 ° 25 ° 30 ~ 25 ~ 25 ~ 30 * 30 °
5:' and 30 °
"t" 1-68 3.45 4"90 3-55 2'56 2-72 2"62 1-98 10"10 4.27 4"69
p >0.10 0.025-0"05 0"01 0.025 0"05-0"10 0.05-0.10 0.05-0.10 >0.10 0'001 0.025 0"01
In addition to the brain lipid analyses a note was also made of the relative amount of lipid in the livers of the same fish. It was observed that in contrast to the results for brain, the lipid content of the liver tended to decrease with a decrease in the acclimation temperature. T h e lipid content of the livers of fish acclimated to 5, 15, 25 and 30°C were 1.76, 2.76, 3.04 and 3"69 per cent respectively. DISCUSSION T h e preceding results are in general agreement with previous work that acclimation of animals to low temperatures leads to an increase in the degree of unsaturation of carcass fat. However, the present study is more comprehensive in that the use of G L C rather than the non-specific iodine number has made it clear that the changes, at least in the brain, involve predominantly the polyunsaturated fatty acids of twenty carbons or more. Furthermore, the observation that the amount of lipid in the brain was higher at lower acclimation temperatures, whereas the opposite trend was shown in the liver, is a clear indication that in order to elucidate the mechanisms involved in the complex process of acclimation, the roles of individual systems must be studied. I f a relationship between changes in lipid fatty acid composition and temperature acclimation exists, it should be possible to assign to lipids a definite role in the process. Since, in contrast to previous work, this study was confined to the lipids
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of the central nervous system, in which it had previously been shown that modifications occurred under identical acclimation conditions (Roots & Prosser, 1962), it is possible to make some suggestions regarding the functions of lipids in the nervous system and the necessity for their alteration in the acclimated animal. The following hypothesis requires two assumptions for its subsequent development. First, the surface of the neuron is considered in terms of a lyotropic mesomorphic or liquid-crystalline system. Furthermore, the ion movements taking place in nervous activity are considered as secondary to electronic conduction and developed potentials due, not to ionic diffusion alone, but to redox potentials and semi-conductivity phenomena. The surface of the neuron is considered to be a lipoprotein liquid-crystalline system dependent for its integrity on the composition of the intra- and extracellular milieux. The physical properties of this system will, to a considerable extent, be a function of those of the fatty acid residues of the constituent phospholipids. These properties will be determined by the geometry, degree of unsaturation, double bond position, degree of chain branching and presence of hydroxyl groups in the fatty acids as well as by the isomeric and polymorphic forms of the phospholipids. A resting potential across such a system may be considered as an asymmetric barrier potential developed by selective ion binding such that an electron excess develops on the inside. Any event which altered conditions on either side would lead to a shift in the membrane potential, The arrival of quanta of acetylcholine from the presynaptic terminal provides one such event. Molecules of acetylcholine by accepting electrons, probably by "charge transfer" from the postsynaptic structure, would cause a shift in the membrane potential. The resultant change in the electric field influencing the "membrane" may trigger the following sequence of events: 1. The release of ions from their binding sites. 2. Conformational changes in the constituents of the lipoprotein system leading to a phase transition. From observations by Luzzati & Husson (1962) and Stoeckenius (1962) on liquid-crystalline states of lipid-water systems it may be deduced that such a transition may be to the crystalline-like coagel state. 3. Increased permeability of the matrix to ions such as Na +. If the local disturbances at the postsynaptic membrane reach a threshold value, the current may be propagated in two ways, either by electronic resonance through molecules containing suitable double bond systems or electronic conduction through the lipoprotein matrix. Once effectively triggered, the electron flow would lead to further increased ion mobility followed by new binding states of the ions on both sides, depolarization and eventually complete reversal of membrane polarity. The system may be visualized as being coupled to ATP hydrolysis brought about as a consequence of electron and ion movements across the "membrane". Mitchell (1961) has recently shown in detail how such coupling between transport and metabolism may occur. The effective functioning of the above system is dependent on the maintenance of the liquid-crystalline state of the resting membrane which is, in turn, dependent on the temperature and on the concentration and nature of the lipid residues of the
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lipoprotein. A particular fatty acid composition and distribution at a specific temperature may thus provide an appropriate solid-liquid ratio, degree of expansibility and hydrophilic properties necessary for the maintenance of a liquidcrystalline system which will undergo a suitable phase transition when influenced by an electrical event. However, a decrease in the environmental temperature of the same system would cause a decrease in thermal agitation and a consequent increase in the lateral attractions of molecules producing either an undesirable phase transition or an inability to undergo a particular transition. Since double bonds attract more water than do saturated linkages, and the maintenance of a liquid-crystalline state is highly dependent on the degree of hydration at a particular temperature, an increase in the degree of unsaturation of the lipids presents a means for maintaining the system near the critical point of a phase transition. An increase in the concentration of lipid may also be necessary for the maintenance of this particular state. If the environmental temperature of the same system was increased, the consequent increase in thermal agitation might lead to a breakdown of the lateral attractions between molecules and disruption of the phase. In this case additional cohesiveness may be achieved by a relative decrease in the number of unsaturated linkages, the consequent water loss leading to a less expanded state of the phase. It follows that if the environmental temperature of the proposed system is changed, adjustments in the structure of the lipoprotein must also facilitate the maintenance of the necessary features for electronic conduction, such as hydrogen bonding and suitable systems for electronic resonance. If a poikilotherm is to accommodate either a lowering or raising of its environmental temperature, it must possess the ability to control the degree of unsaturation of its cellular lipids. The mechanism by which this is achieved is obscure and may depend on a temperature-sensitive reaction in fatty acid synthesis. Such a reaction has recently been suggested as the primary cause of the effect of temperature on the fatty acid composition of certain micro-organisms (Bishop & Still, 1963). Since the cerebrosides and other myelin lipids do contain considerable quantities of non-hydroxylated fatty acids, the results obtained in this study may reflect changes occurring in myelin as well as in the lipids of neuronal and glial elements. However, the theories postulated were confined to changes in the neuronal lipids since it seems reasonable to assume that appropriate structural adjustments to environmental change are made in the myelin lipids which are, in effect, derivatives of the Schwann cells and glial elements. The complete elucidation of the mechanisms involved in acclimation may, however, only be obtained by detailed studies of the molecular architecture of the specific cells and subcellular elements involved. REFERENCES BISHOP D. G. • STILL J. L. (1963) Fatty acid metabolism in Serratia marcescens. IV. T h e effect of temperature on fatty acid composition. J. Lipid Res. 4, 87-90. F.*WeETT D. W. & LYMAN C. P. (1954) T h e effect of low environmental temperature of depot fat in relation to hibernation. J. Physiol. 126, 235-237.
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FOLCH J . , LEES M. • SLOANE STANLEY G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. 07. Biol. Chem. 226, 497-509. FRAENKELG. & HOPF H. S. (1940) T h e physiological action of abnormally high temperatures on poikilothermic animals. I. Temperature adaptation and the degree of saturation of the phosphatides. Biochem.07. 34, 1085-1092. HOAR W. S. & COTTLE M. K. (1952) Some effects of temperature acclimatization on the chemical constitution of goldfish tissues. Canad. 07. Zool. 30, 49-54. HOUSE H. L., RIORDAN D. F. & BARLOWJ. S. (1958) Effects of thermal conditioning and of degree of saturation of dietary lipids on resistance of an insect to high temperature. Canad. 07. Zool. 36, 629-632. JOHNSTON P. V., KOPACZYKK. C. & KUMMEROWF. A. (1961) Effect of pyridoxine deficiency on fatty acid composition of carcass and brain lipids in the rat. 07. Nutr. 74, 96-102. JOHNSTON P. V. & KUMMEROWF. A. (1960) Gas-liquid chromatography of methyl esters of fatty acids from human and chicken brain lipids. Proc. Soc. Exp. Biol. Med. 104, 201-205. KISHIMOTOY. & RADIN N. S. (1959) Isolation and determination methods for brain cerebrosides, hydroxy fatty acids and unsaturated and saturated fatty acids. 07. Lipid Res. I, 72-78. LUZZATI V. & HUSSON F. (1962) The structure of liquid-crystalline phases of lipid-water systems. 07. Cell. Biol. 12, 207-219. MITCHELL P. (1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature, Lond. 191, 144-148. RooTs B. I. & PROSSER C. L. (1962) Temperature acclimation and the nervous system in fish. 07. exp. Biol. 39, 617-629. SNEDECOR G. W. (1956) Statistical Methods, 5th ed. Iowa State College, Ames, Iowa. STOECKENIUS W. (1962) Some electron microscopical observations on liquid-crystalline phases in lipid-water systems. 07. Cell. Biol. 12, 221-229. WOODFORD F. P. & VAN GENT C. M. (1960) Gas-liquid chromatography of fatty acid methyl esters: T h e "carbon n u m b e r " as a parameter for comparison of columns. J. Lipid Res. 1, 188-189.