Neurochemical Research (2) 379-393 (1977)
A C Y L A N D A L K E N Y L GROUP COMPOSITION OF BRAIN S U B C E L L U L A R FRACTIONS OF GOLDFISH (Carassius auratus L.) ACCLIMATED TO D I F F E R E N T ENVIRONMENTAL TEMPERATURES DANIEL P. SELIVONCHICK, 1"2 PATRICIA V. JOHNSTON, 3 AND BETTY
I. ROOTS 1
Department of Zoology and Erindale College University of Toronto Mississauga, Ontario, Canada LSL 1C6 Department of Food Science University of lUinois Urbana, Illinois 6180l
Accepted January 25, 1977
Goldfish were acclimated to 5, 15, and 30~ and the acyl group composition of choline phosphoglycerides (CPG) and ethanolamine phosphoglycerides (EPG) from whole brain and brain subcellular particles was examined. With the exception of synaptosomal CPG, the acyl group composition of CPG from whole brain and subcellular particles, including myelin, from cold-acclimated fish showed little response to the change in environmental temperature. Those changes that did occur were consistent with the expected trend toward a higher degree of unsaturation of the CPG acyl groups in fish acclimated to 5~ The acyl group composition of CPG from synaptosomes of the cold-acclimated fish did, however, differ markedly in having a reduced unsaturation index (U.I.) and unsaturated: saturated fatty acid ratio (UFA: SFA) which was caused mainly by the decrease in 22:6 n-3 content. In contrast, changes in the acyl group composition of EPG on cold acclimation were greater than those observed in any 1 Department of Zoology and Erindale College, University of Toronto, Mississauga, Ontario, Canada L5L IC6. 2 Present address: Department of Food Science and Technology, Oregon State University, Corvallis, Oregon 97331. 3 Department of Food Science, University of Illinois, Urbana, Illinois 61801.
379 This journal is copyrighted by Plenum. Each article is available for $7.50 from Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011.
380
SELIVONCHICK ET AL.
CPG fraction. The generally expected trend toward greater unsaturation was observed only in mitochondrial and myelin EPG. Moreover, in all fractions the amount of 22:6 n-3 in EPG was lower at decreased environmental temperatures. In the synaptosomal and microsomal EPG, the reduction in 22:6 n-3 was such that a markedly reduced U.I. was obtained. It is suggested that two compensatory mechanisms maintain the necessary degree of membrane permeability and fluidity in order to prevent transition to a crystalline state at lower temperatures.
INTRODUCTION Cold acclimation in poikilotherms has been associated invariably with an increased degree of unsaturation of the fatty acid residues of phospholipids. The fatty acids involved show specificity with respect to the species and tissue, but the overall trend is considered to be unambiguous. The most studied species in this regard is the goldfish Carassius auratus L. In this species, as in others, the overall trend to greater unsaturation at lower temperatures pertains and has been established in several tissues including the brain (1-8). With the exception of a decreased plasmalogen content in coldacclimated animals (5,7), however, the relative phosphoglyceride composition of goldfish brain does not change (6). The response to cold acclimation has been interpreted as a compensatory mechanism by which lipids characterized by lower melting points and greater expansibility in monomolecular films are laid down in order to offset the increase in molecular cohesion at low temperatures. In other words, the mechanism is visualized as necessary to maintain membrane permeability and fluidity, and to prevent transition from a liquid-crystalline to a crystalline phase within the environmental temperature range (2,5,6,9,10). In addition, it has been suggested that the changes in the nature of membrane lipids not only maintain fluidity but also may modulate the activity of membrane-bound enzymes (5,6,11). These explanations are, however, based on extrapolation from artificial membranes of a relatively simple nature and on evidence that, in some species, membrane-based enzymes exhibit a requirement for specific degree of unsaturation of membrane lipids (12-17). Only in the case of the mitochondria of goldfish gill tissue (18) have studies on the degree of fatty acid unsaturation been extended to the organelle level. Studies on brain subceUular fractions may prove to be of particular interest, since brain possesses both the usual subcellular fractions and the highly specialized myelin membrane and synaptosomes. We have examined the two major phospholipids of these brain subcellular fractions with the view that the particular changes taking
ENVIRONMENTAL TEMPERATURE AND BRAIN LIPIDS
381
place in each functional unit may provide further insight regarding the types of changes in membrane-based lipids that are essential to the maintenance of their efficient function.
EXPERIMENTAL PROCEDURE
Source, Maintenance, and Acclimation ofFish Goldfish (Carassius auratus L.), 5-6 in. in total length, were obtained from a pond in Collingwood, Ontario, and from Hartz Mountain P~t Supplies Ltd., Toronto, Ontario. Upon arrival in the laboratory, the fish were placed in holding tanks at 10~ and kept at this temperature for at least 30 days before being transferred to tanks at a different temperature. The fish were divided into three groups, acclimated to 5, 15, or 30~ respectively, for a period of 49 days. They were kept under conditions of constant length of day (12 h of light and 12 h of darkness) and were fed Purina Trout Chow. Fish at 30~ were fed twice a day, those at 15~ once a day, and those at 5~ once every other day, all ad libitum.
Preparation of Subcellular and Myelin Fractions Subcellular fractions were prepared from pools of five brains from goldfish kept at each acclimation temperature, by the method of Sun and Sun (19). The purity of each fraction was assessed by electron microscopy. The fractions were quick-frozen and stored at -70~ until analyzed. Mitochondrial, microsomal, and synaptosomal fractions were found to be virtually free of contamination from other membrane types. Myelin was isolated from fresh brains and frozen spinal cords by the method of Agrawal et al. (20). The myelin fractions were obtained from pools of 14 brains and 14 spinal cords for each acclimation temperature. The isolated myelin was lyophilized and stored at -70~ until analyzed. Aliquots of the myelin fractions were assessed for purity by electron microscopy as described elsewhere (2 I).
Extraction of Lipids A l-g sample of the diet was homogenized with 20 vol of chloroform-methanol (2: 1, vol/ vot) and filtered. The residue was collected from the filter paper and extracted two more times with 10 vol of chloroform-methanol (2:1, vol/vol). The extracts were pooled and washed with 0.2 vol of water, and the lower phase collected and dried in a rotary vacuum evaporator. Aliquots of the diet lipids were removed and transesterified for analysis by gas-liquid chromatography (GLC). The acyl group composition of the diet is shown in Table I. Lipid extracts from whole brain were prepared as previously described (21). Myelin lipids were extracted according to Autilio et al. (22) and from subcellular fractions by the method of Sun and Horrocks (23). The protein-free lipid solutions were taken to dryness under nitrogen. After a thorough flushing with nitrogen, the samples were dissolved in chloroform-methanol (2:! vol/vol) and stored at -70~ until analyzed.
382
SELIVONCHICK ET AL.
Analysis of Acyl and Alkenyl Groups Methyl esters were prepared from EPG and CPG. The individual phospholipids were separated by two-dimensional TLC as previously described (21). After development in the second dimension, the lipids were visualized by spraying with 2' ,7'-dichlorofluorescein and viewing under ultraviolet light. The EPG and CPG spots were removed and subjected to methanolysis by heating in 4% sulfuric acid in methanol in sealed vials at 85-90~ for 90 min. The methyl esters and dimethyl acetals formed were extracted with hexane, washed with 5% sodium bicarbonate, and then dried over anyhdrous sodium sulfate. The fractions were purified, and the dimethyl acetals separated from the methyl esters on thin-layer plates (silica gel G) developed in benzene. Argentation TLC was performed on plates coated with a 0.5-ram-thick slurry of 12% (wt/ wt) silver nitrate in water with silica gel G (24). The plates were air dried and stored in the dark until used. Prior to use, the plates were activated for 30 rain at 120~ Methyl esters were applied as a narrow band, and the plates developed for 15 cm in a solvent system of hexane-diethyl ether-glacial acetic acid (90: 10: I, vol/vol/vol). The saturated band was removed and eluted with moist diethyl ether. The sample was then analyzed by GLC. This analysis was performed to verify the absence of long-chain saturated fatty acids with an equivalent chain length (ECL) corresponding to 22:6 n-3.
Gas Liquid Chromotograhpy The EPG and CPG fatty acid patterns were determined with a Hewlett-Packard 7026A research gas chromatograph. The samples were injected onto a 6-ft glass column (3 mm i.d.) packed with 15% ethylene glycol succinate on 80/100 mesh Gas Chrom P (Hi-Eff2BP, Applied Science Laboratories, Inc., State College, Pa.). The operating column temperature for methyl ester analysis was either programmed to rise from 140 to 180~ or was kept isothermal at 180~ Dimethyl acetals were analyzed at 150~ Peaks were identified by comparison of relative retention times with those of standards obtained from Supelco, Inc., and by determination of equivalent chain length according to Hofstetter et al. (25). Peak areas were determiend by triangulation or with a Hewlett-Packard 3380A integrator. Quantitative results with fatty acid standard F (NIH Mixture) differed from the stated composition by a relative error of less than 6% for major components (> 10% of mixture) and less than 5% for minor components ( < 10% of mixture). These values for the percent relative error are the means of six determinations.
RESULTS
Effect of Temperature on the Acyl Group Composition of CPG and EPG from Whole Brain The acyl group composition of CPG and EPG from whole brain lipid of fish acclimated to 5, 15, and 30~ is shown in Table I. The effect of cold acclimation on the CPG acyl groups is consistent with the classic picture; namely, there is a decided trend toward a higher degree of unsaturation at 5~ This higher degree of unsaturation (Table 1) is
383
E N V I R O N M E N T A L T E M P E R A T U R E A N D B R A I N LIPIDS
TABLE
I
COMPOSITION OF THE CONSTITUENT FATTY ACIDS (WT ~b) OF THE DIET AND OF CHOLINE PHOSPHOGLYCERIDES ( C P G ) AND ETHANOLAMINE PHOSPHOGLYCERIDES(EPG) PREPARED FROM BRAINS a OF GOLDFISH ACCLIMATED TO DIFFERENT ENVIRONMENTAL TEMPERATURES CPG Acyl group
Diet
5~
14:0 16:0 16:In-7 18:0 18:In-9 18:2n-6 20:ln-9 20:2n-9 + n-6 20:3n-6 20:4n-6 20:5n-6 22:4n-6 22:5n-6 + n-3 22:6n-3 U.I. b U F A : SFA C
5.2 19.6 4.0 5.8 21.6 23.2 3.2 1.0 0.2 0.9 7.3 -1.0 7.1
. 24.3 8.5 7.9 24.7 1,3 3.4 1.3 1.5 6.9 1.2 1.6 0.6 17.5 194 2.1
EPG
15~ .
30 ~ .
29.7 8.3 7.0 25.8 1.2 3.8 1.1 1.3 6.0 0.9 1.1 0.4 13.6 163 1.7
. 32.2 6.6 8.0 29.7 1.1 2.6 0.7 1.2 3.9 0.4 1,1 tr a 12.9 146 1.5
5~ . 8.1 6.2 19.9 16.3 1.5 3.7 2.0 1.9 13.2 2.3 1.6 1.7 32.0 310 4.6
15~
30 ~
7,4 5.2 11.2 16.5 0.9 3.0 2,0 1.8 11.3 1.6 1.5 1.0 36.8 321 4.4
8.9 4.4 15.2 16.6 0.6 1.9 1,2 1.5 8.9 0.8 1.2 0.6 38.7 311 3.2
.
~Lipid extracts were prepared separately from two brains from each acclimation tempera-
ture; the results are the average of four determinations (duplicates on each extract). b U.I. = unsaturation index, defined as E (number of double bonds in each fatty acid) • (tool % of each fatty acid). c U F A : S F A = ratio of the sum of unsaturated fatty acids to the sum of saturated fatty
acids. ,1 tr = trace.
caused mainly by increases in 20:4 n-6 and 22:6 n-3 and is reflected in the higher unsaturation index (U.I.) and higher ratio of unsaturated to saturated fatty acids (UFA: SFA). In contrast to the increase in highly unsaturated fatty acids 20:4 n-6 and 22:3 n-6, the major monoenoic fatty acid 18:1 n-9 is decreased at the lower temperature. The acyl group composition of CPG from fish acclimated to the intermediate temperature shows a pattern that lies between those of the two extremes (Table
I). The composition of EPG from cold-acclimated fish is consistent with previous findings with respect to the increased levels of the polyunsaturated fatty acids (PUFA) 20:4 n-6 and 20:5 n-6, but not with respect to 22:6 n-3, which decreased by 17.3% at 5~ (Table I). However, due to
384
S E L I V O N C H I C K E T AL.
the relatively small difference in the major monoenoic 9 at the two temperatures, there was no change in the UFA: SFA was only slightly elevated at the lower composition of EPG from fish acclimated to 15~ general changes (Table I).
fatty acids 18:1 nU.I., and the ratio temperature. The shows the same
Effect of Temperature on the Acyl Group Composition of CPG and EPG of Brain Subcellular Fractions and Brain and Spinal Cord Myelin Cold acclimation had markedly different effects on the acyl group composition of the phospholipids from brain subcellular fractions and brain and spinal cord myelin. The composition of the CPG from the mitochondrial and microsomal fraction showed little change with temperature. While slight increases in both the U.I. and U F A : S F A were noted, this was the result of increases in 16:1 in both the mitochondrial and microsomal CPG and of decreases in 16:0 and 18:0 in the mitochondrial and in 18:0 in the microsomal CPG (Table II). In contrast, the CPG from the synaptosomes showed a marked decrease in the U.I. of its acyl groups and a decrease in the UFA: SFA ratio. This unexpected finding was largely caused by a 26.4% decrease in the amount of 22:6 n-3 in the CPG from cold-acclimated fish. In addition, there was an increase in the amount of 16:0 and a decrease in 20:4 n-6. The composition of CPG acyl groups in synaptosomes from fish acclimated to 15~ closely resembled that seen in 30~ fish. Both brain and spinal cord myelin CPG showed a trend toward greater unsaturation at the lower environmental temperatures, largely caused by an increase in 20:4 n-6. No striking differences were found in the acyl group composition of CPG from the two sources of myelin. In keeping with the effects seen in whole brain EPG, there were markedly different changes in the acyl group composition of EPG from subcellular fractions of brain and spinal cord myelin (Table III). The EPG from mitochondria and both myelin fractions from cold-acclimated fish had a greater degree of unsaturation, and this is reflected in the increased U.I. and U F A : S F A ratio. However, in all fractions the amount of 22:6 n-3 was decreased on acclimation to lower temperatures, and the trend of this fatty acid to decrease with a decrease in environmental temperature was also observed in EPG of fish acclimated to 15~ The increase in unsaturation of mitochondrial EPG acyl groups was caused by a combination of depressed levels of 16:0 and 18:0 and increased levels of 18:1 n-9 and 20:4 n-6 at the lower temperatures. The
385
ENVIRONMENTAL TEMPERATURE A N D BRAIN LIPIDS
A C Y L A N D A L K E N Y L GROUP COMPOSITION OF BRAIN S U B C E L L U L A R FRACTIONS OF GOLDFISH (Carassius auratus L.) ACCLIMATED TO D I F F E R E N T ENVIRONMENTAL TEMPERATURES DANIEL P. SELIVONCHICK, 1"2 PATRICIA V. JOHNSTON, 3 AND BETTY
I. ROOTS 1
Department of Zoology and Erindale College University of Toronto Mississauga, Ontario, Canada LSL 1C6 Department of Food Science University of lUinois Urbana, Illinois 6180l
Accepted January 25, 1977
Goldfish were acclimated to 5, 15, and 30~ and the acyl group composition of choline phosphoglycerides (CPG) and ethanolamine phosphoglycerides (EPG) from whole brain and brain subcellular particles was examined. With the exception of synaptosomal CPG, the acyl group composition of CPG from whole brain and subcellular particles, including myelin, from cold-acclimated fish showed little response to the change in environmental temperature. Those changes that did occur were consistent with the expected trend toward a higher degree of unsaturation of the CPG acyl groups in fish acclimated to 5~ The acyl group composition of CPG from synaptosomes of the cold-acclimated fish did, however, differ markedly in having a reduced unsaturation index (U.I.) and unsaturated: saturated fatty acid ratio (UFA: SFA) which was caused mainly by the decrease in 22:6 n-3 content. In contrast, changes in the acyl group composition of EPG on cold acclimation were greater than those observed in any 1 Department of Zoology and Erindale College, University of Toronto, Mississauga, Ontario, Canada L5L IC6. 2 Present address: Department of Food Science and Technology, Oregon State University, Corvallis, Oregon 97331. 3 Department of Food Science, University of Illinois, Urbana, Illinois 61801.
379 This journal is copyrighted by Plenum. Each article is available for $7.50 from Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011.
380
SELIVONCHICK ET AL.
CPG fraction. The generally expected trend toward greater unsaturation was observed only in mitochondrial and myelin EPG. Moreover, in all fractions the amount of 22:6 n-3 in EPG was lower at decreased environmental temperatures. In the synaptosomal and microsomal EPG, the reduction in 22:6 n-3 was such that a markedly reduced U.I. was obtained. It is suggested that two compensatory mechanisms maintain the necessary degree of membrane permeability and fluidity in order to prevent transition to a crystalline state at lower temperatures.
INTRODUCTION Cold acclimation in poikilotherms has been associated invariably with an increased degree of unsaturation of the fatty acid residues of phospholipids. The fatty acids involved show specificity with respect to the species and tissue, but the overall trend is considered to be unambiguous. The most studied species in this regard is the goldfish Carassius auratus L. In this species, as in others, the overall trend to greater unsaturation at lower temperatures pertains and has been established in several tissues including the brain (1-8). With the exception of a decreased plasmalogen content in coldacclimated animals (5,7), however, the relative phosphoglyceride composition of goldfish brain does not change (6). The response to cold acclimation has been interpreted as a compensatory mechanism by which lipids characterized by lower melting points and greater expansibility in monomolecular films are laid down in order to offset the increase in molecular cohesion at low temperatures. In other words, the mechanism is visualized as necessary to maintain membrane permeability and fluidity, and to prevent transition from a liquid-crystalline to a crystalline phase within the environmental temperature range (2,5,6,9,10). In addition, it has been suggested that the changes in the nature of membrane lipids not only maintain fluidity but also may modulate the activity of membrane-bound enzymes (5,6,11). These explanations are, however, based on extrapolation from artificial membranes of a relatively simple nature and on evidence that, in some species, membrane-based enzymes exhibit a requirement for specific degree of unsaturation of membrane lipids (12-17). Only in the case of the mitochondria of goldfish gill tissue (18) have studies on the degree of fatty acid unsaturation been extended to the organelle level. Studies on brain subceUular fractions may prove to be of particular interest, since brain possesses both the usual subcellular fractions and the highly specialized myelin membrane and synaptosomes. We have examined the two major phospholipids of these brain subcellular fractions with the view that the particular changes taking
ENVIRONMENTAL TEMPERATURE AND BRAIN LIPIDS
381
place in each functional unit may provide further insight regarding the types of changes in membrane-based lipids that are essential to the maintenance of their efficient function.
EXPERIMENTAL PROCEDURE
Source, Maintenance, and Acclimation ofFish Goldfish (Carassius auratus L.), 5-6 in. in total length, were obtained from a pond in Collingwood, Ontario, and from Hartz Mountain P~t Supplies Ltd., Toronto, Ontario. Upon arrival in the laboratory, the fish were placed in holding tanks at 10~ and kept at this temperature for at least 30 days before being transferred to tanks at a different temperature. The fish were divided into three groups, acclimated to 5, 15, or 30~ respectively, for a period of 49 days. They were kept under conditions of constant length of day (12 h of light and 12 h of darkness) and were fed Purina Trout Chow. Fish at 30~ were fed twice a day, those at 15~ once a day, and those at 5~ once every other day, all ad libitum.
Preparation of Subcellular and Myelin Fractions Subcellular fractions were prepared from pools of five brains from goldfish kept at each acclimation temperature, by the method of Sun and Sun (19). The purity of each fraction was assessed by electron microscopy. The fractions were quick-frozen and stored at -70~ until analyzed. Mitochondrial, microsomal, and synaptosomal fractions were found to be virtually free of contamination from other membrane types. Myelin was isolated from fresh brains and frozen spinal cords by the method of Agrawal et al. (20). The myelin fractions were obtained from pools of 14 brains and 14 spinal cords for each acclimation temperature. The isolated myelin was lyophilized and stored at -70~ until analyzed. Aliquots of the myelin fractions were assessed for purity by electron microscopy as described elsewhere (2 I).
Extraction of Lipids A l-g sample of the diet was homogenized with 20 vol of chloroform-methanol (2: 1, vol/ vot) and filtered. The residue was collected from the filter paper and extracted two more times with 10 vol of chloroform-methanol (2:1, vol/vol). The extracts were pooled and washed with 0.2 vol of water, and the lower phase collected and dried in a rotary vacuum evaporator. Aliquots of the diet lipids were removed and transesterified for analysis by gas-liquid chromatography (GLC). The acyl group composition of the diet is shown in Table I. Lipid extracts from whole brain were prepared as previously described (21). Myelin lipids were extracted according to Autilio et al. (22) and from subcellular fractions by the method of Sun and Horrocks (23). The protein-free lipid solutions were taken to dryness under nitrogen. After a thorough flushing with nitrogen, the samples were dissolved in chloroform-methanol (2:! vol/vol) and stored at -70~ until analyzed.
382
SELIVONCHICK ET AL.
Analysis of Acyl and Alkenyl Groups Methyl esters were prepared from EPG and CPG. The individual phospholipids were separated by two-dimensional TLC as previously described (21). After development in the second dimension, the lipids were visualized by spraying with 2' ,7'-dichlorofluorescein and viewing under ultraviolet light. The EPG and CPG spots were removed and subjected to methanolysis by heating in 4% sulfuric acid in methanol in sealed vials at 85-90~ for 90 min. The methyl esters and dimethyl acetals formed were extracted with hexane, washed with 5% sodium bicarbonate, and then dried over anyhdrous sodium sulfate. The fractions were purified, and the dimethyl acetals separated from the methyl esters on thin-layer plates (silica gel G) developed in benzene. Argentation TLC was performed on plates coated with a 0.5-ram-thick slurry of 12% (wt/ wt) silver nitrate in water with silica gel G (24). The plates were air dried and stored in the dark until used. Prior to use, the plates were activated for 30 rain at 120~ Methyl esters were applied as a narrow band, and the plates developed for 15 cm in a solvent system of hexane-diethyl ether-glacial acetic acid (90: 10: I, vol/vol/vol). The saturated band was removed and eluted with moist diethyl ether. The sample was then analyzed by GLC. This analysis was performed to verify the absence of long-chain saturated fatty acids with an equivalent chain length (ECL) corresponding to 22:6 n-3.
Gas Liquid Chromotograhpy The EPG and CPG fatty acid patterns were determined with a Hewlett-Packard 7026A research gas chromatograph. The samples were injected onto a 6-ft glass column (3 mm i.d.) packed with 15% ethylene glycol succinate on 80/100 mesh Gas Chrom P (Hi-Eff2BP, Applied Science Laboratories, Inc., State College, Pa.). The operating column temperature for methyl ester analysis was either programmed to rise from 140 to 180~ or was kept isothermal at 180~ Dimethyl acetals were analyzed at 150~ Peaks were identified by comparison of relative retention times with those of standards obtained from Supelco, Inc., and by determination of equivalent chain length according to Hofstetter et al. (25). Peak areas were determiend by triangulation or with a Hewlett-Packard 3380A integrator. Quantitative results with fatty acid standard F (NIH Mixture) differed from the stated composition by a relative error of less than 6% for major components (> 10% of mixture) and less than 5% for minor components ( < 10% of mixture). These values for the percent relative error are the means of six determinations.
RESULTS
Effect of Temperature on the Acyl Group Composition of CPG and EPG from Whole Brain The acyl group composition of CPG and EPG from whole brain lipid of fish acclimated to 5, 15, and 30~ is shown in Table I. The effect of cold acclimation on the CPG acyl groups is consistent with the classic picture; namely, there is a decided trend toward a higher degree of unsaturation at 5~ This higher degree of unsaturation (Table 1) is
383
E N V I R O N M E N T A L T E M P E R A T U R E A N D B R A I N LIPIDS
TABLE
I
COMPOSITION OF THE CONSTITUENT FATTY ACIDS (WT ~b) OF THE DIET AND OF CHOLINE PHOSPHOGLYCERIDES ( C P G ) AND ETHANOLAMINE PHOSPHOGLYCERIDES(EPG) PREPARED FROM BRAINS a OF GOLDFISH ACCLIMATED TO DIFFERENT ENVIRONMENTAL TEMPERATURES CPG Acyl group
Diet
5~
14:0 16:0 16:In-7 18:0 18:In-9 18:2n-6 20:ln-9 20:2n-9 + n-6 20:3n-6 20:4n-6 20:5n-6 22:4n-6 22:5n-6 + n-3 22:6n-3 U.I. b U F A : SFA C
5.2 19.6 4.0 5.8 21.6 23.2 3.2 1.0 0.2 0.9 7.3 -1.0 7.1
. 24.3 8.5 7.9 24.7 1,3 3.4 1.3 1.5 6.9 1.2 1.6 0.6 17.5 194 2.1
EPG
15~ .
30 ~ .
29.7 8.3 7.0 25.8 1.2 3.8 1.1 1.3 6.0 0.9 1.1 0.4 13.6 163 1.7
. 32.2 6.6 8.0 29.7 1.1 2.6 0.7 1.2 3.9 0.4 1,1 tr a 12.9 146 1.5
5~ . 8.1 6.2 19.9 16.3 1.5 3.7 2.0 1.9 13.2 2.3 1.6 1.7 32.0 310 4.6
15~
30 ~
7,4 5.2 11.2 16.5 0.9 3.0 2,0 1.8 11.3 1.6 1.5 1.0 36.8 321 4.4
8.9 4.4 15.2 16.6 0.6 1.9 1,2 1.5 8.9 0.8 1.2 0.6 38.7 311 3.2
.
~Lipid extracts were prepared separately from two brains from each acclimation tempera-
ture; the results are the average of four determinations (duplicates on each extract). b U.I. = unsaturation index, defined as E (number of double bonds in each fatty acid) • (tool % of each fatty acid). c U F A : S F A = ratio of the sum of unsaturated fatty acids to the sum of saturated fatty
acids. ,1 tr = trace.
caused mainly by increases in 20:4 n-6 and 22:6 n-3 and is reflected in the higher unsaturation index (U.I.) and higher ratio of unsaturated to saturated fatty acids (UFA: SFA). In contrast to the increase in highly unsaturated fatty acids 20:4 n-6 and 22:3 n-6, the major monoenoic fatty acid 18:1 n-9 is decreased at the lower temperature. The acyl group composition of CPG from fish acclimated to the intermediate temperature shows a pattern that lies between those of the two extremes (Table
I). The composition of EPG from cold-acclimated fish is consistent with previous findings with respect to the increased levels of the polyunsaturated fatty acids (PUFA) 20:4 n-6 and 20:5 n-6, but not with respect to 22:6 n-3, which decreased by 17.3% at 5~ (Table I). However, due to
384
S E L I V O N C H I C K E T AL.
the relatively small difference in the major monoenoic 9 at the two temperatures, there was no change in the UFA: SFA was only slightly elevated at the lower composition of EPG from fish acclimated to 15~ general changes (Table I).
fatty acids 18:1 nU.I., and the ratio temperature. The shows the same
Effect of Temperature on the Acyl Group Composition of CPG and EPG of Brain Subcellular Fractions and Brain and Spinal Cord Myelin Cold acclimation had markedly different effects on the acyl group composition of the phospholipids from brain subcellular fractions and brain and spinal cord myelin. The composition of the CPG from the mitochondrial and microsomal fraction showed little change with temperature. While slight increases in both the U.I. and U F A : S F A were noted, this was the result of increases in 16:1 in both the mitochondrial and microsomal CPG and of decreases in 16:0 and 18:0 in the mitochondrial and in 18:0 in the microsomal CPG (Table II). In contrast, the CPG from the synaptosomes showed a marked decrease in the U.I. of its acyl groups and a decrease in the UFA: SFA ratio. This unexpected finding was largely caused by a 26.4% decrease in the amount of 22:6 n-3 in the CPG from cold-acclimated fish. In addition, there was an increase in the amount of 16:0 and a decrease in 20:4 n-6. The composition of CPG acyl groups in synaptosomes from fish acclimated to 15~ closely resembled that seen in 30~ fish. Both brain and spinal cord myelin CPG showed a trend toward greater unsaturation at the lower environmental temperatures, largely caused by an increase in 20:4 n-6. No striking differences were found in the acyl group composition of CPG from the two sources of myelin. In keeping with the effects seen in whole brain EPG, there were markedly different changes in the acyl group composition of EPG from subcellular fractions of brain and spinal cord myelin (Table III). The EPG from mitochondria and both myelin fractions from cold-acclimated fish had a greater degree of unsaturation, and this is reflected in the increased U.I. and U F A : S F A ratio. However, in all fractions the amount of 22:6 n-3 was decreased on acclimation to lower temperatures, and the trend of this fatty acid to decrease with a decrease in environmental temperature was also observed in EPG of fish acclimated to 15~ The increase in unsaturation of mitochondrial EPG acyl groups was caused by a combination of depressed levels of 16:0 and 18:0 and increased levels of 18:1 n-9 and 20:4 n-6 at the lower temperatures. The
385
ENVIRONMENTAL TEMPERATURE A N D BRAIN LIPIDS
