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Haematological values and fatty acid composition of erythrocyte phospholipids in cod (Gadus morhua) fed at different water temperatures
Aquaculture, 79 (1989) 137-144 Elsevier Science Publishers B.V., Amsterdam -Printed
137 in The Netherlands
Haematological Values and Fatty Acid Composition of Erythrocyte Phospholipids in Cod (Gadus morhua) Fed at Different Water Temperatures 0YVIND LIE, EINAR LIED and GEORG LAMBERTSEN Institute of Nutrition, Directorate of Fisheries, P.0. Box 1900, N-5024 Bergen (Norway)
ABSTRACT Lie, O., Lied, E. and Lambertsen, G., 1989. Haematological values and fatty acid composition of erythrocyte phospholipids in cod (Godus morhua) fed at different water temperatures. Aquaculture, 79: 137-144. Haematological values and fatty acid composition of erythrocyte phospholipids were determined in cod (Gadus morhua) which had been fed at 812 and 16” C. The number of erythrocytes and the values of haematocrit and haemoglobin were highest at 16 OC whereas the mean cell volume and mean cell haemoglobin were lowest at this temperature. The mean cell haemoglobin concentration was not affected by water temperature. The fatty acid composition of the main phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE ) , phosphatidylserine (PS) and phosphatidylinositol (PI), in the erythrocytes was determined. The relative levels of both saturated and monoene fatty acids declined with decreasing water temperature and the polyunsaturated fatty acid fraction generally increased.
INTRODUCTION
Fish haematological values undergo seasonal variations concomitant to climatic changes in light and water temperature (H&dig and Hoglund, 1983). The effects of temperature on lipid composition of fish have recently been reviewed by Bell et al. (1986) and Greene and Selivonchick (1987)) showing that the ratio of polyunsaturated to saturated fatty acids in fish membrane lipids increases in response to lowered ambient water temperature. Erythrocyte metabolism and function are dependent on the functionality of the membrane, wherein many of the important metabolic and transport systems of the cells are embedded. We have earlier demonstrated that the fatty acid composition of fish lipids is affected by the fatty acids present in the feed lipid. In the present paper we describe how the fish is able to adjust its lipid composition according to temperature.
0044-8486/89/$03.50
0 1989 Elsevier Science Publishers B.V.
138 MATERIALS AND METHODS
Fish and diets. Cod (mean weight 180 g) were obtained from culture at the Aquaculture Station Austevoll (Institute of Marine Research, Directorate of Fisheries, Norway). Three groups each of 20 fish were kept in aquaria of 175 1 for 9 weeks at 8,12 and 16°C after a gradual temperature acclimatization for 4 weeks. The fish were fed to satiation once a day 5 days a week. The diet was based on squid mantle (91.4% ), capelin oil (4.1% ), dextrinized potato starch (4.3% ) and a mixture (0.2% ) of vitamins and minerals according to Lie et al. (1986). Analytical methods. Blood samples were collected from the ductus cuvieri and treated according to Sandnes et al. (1988). Red blood cell count (RBC ), haematocrit (Hct ) and haemoglobin (Hb ) were determined according to Sandnes et al. (1988). A portion of the blood was centrifuged at 3000 g and the erythrocytes were washed three times in an isotonic saline solution. Total lipid was extracted from washed erythrocytes with chloroform/methanol (2:1, u/u). The four phospholipid groups phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS) and phosphatidylinositol (PI ) were separated by an adsorption HPLC method modified from Geurts van Kessel et al. (1977) (Lie and Lambertsen, to be published). Each phospholipid fraction was saponified and the fatty acids were methylated in 12% borontrifluoride in methanol. The methyl esters were analysed using a Carlo Erba 2900 gas chromatograph equipped with a 50-m CP-sil88 (Chrompack) fused silica capillary column (i.d. 0.32 mm). The fatty acid composition was calculated using an integrator (Shimadzu C-R3A) and peak identification was ascertained by a standard mixture of methyl esters (Nu-Chek Prep., Elysian, MN, U.S.A.). Statistics. The analytical data were evaluated statistically by means of the Student’s t-test. RESULTS AND DISCUSSION
The analytical data presented in this paper relate to blood samples from three groups of cod which were allowed to grow for 9 weeks at 8,12 and 16°C on a moist diet composed of squid mantle and capelin as the protein and lipid source. During this period the cod grew from an average weight of 180 g to about 250 g at 8°C and to about 280 g at 12 and 16°C. Table 1 shows that the fish was able to adjust its blood according to temperature during the growth period; i.e. the number of red blood cells (RBC), the haematocrit (Hct ) and haemoglobin (Hb) were significantly lower at 8” C than at 12 and 16 oC. The mean cell haemoglobin concentration (MCHC ) , however, was not affected by the temperature the fish had been subjected to, whereas
139 TABLE 1 Haematological values (mean k SD, n = 8) in cod held at different water temperatures Significant differences between groups
Temperature ( “C)
16 RBC (~10~~ 1-l) SD Hct (%) SD Hb (g 100 ml-l) SD MCH (x10-6g-‘) SD MCV (x lo-l5 1-l) SD MCHC (g 100 ml-‘) SD
1.31 0.12 28.5 2.4 5.35 0.48 41 4 219 16 18.8 0.7
12
8
16-8
16-12
12-8
1.10
1.03
***
**
0.14 27.9
0.17 25.3
**
-
*
2.7 5.25 0.66 48
2.8 4.80 0.62 47
* **
***
_
**
***
3 256 22 18.8 0.8
6 249 31 19.0 0.9
-
*O.Ol
the mean cell haemoglobin (MCH) an the mean cell volume (MCV) were markedly lower at 16°C than at 8 and 12°C. Adaptation to elevated water temperature has been shown to induce a number of haematological changes (Houston, 1980). Lane (1979) reported a reduction in RBC, Hct and Hb in rainbow trout (SC&O guirdneri) during the period from October to March, but these parameters were not significantly correlated to water temperature. Similarly, Munkittrick and Leatherland (1983) reported a significant increase in Hct in goldfish (Carassius aurutus L. ) with increased water temperature. An increase in MCH and MCHC was reported by Lane (1979) in rainbow trout, and was interpreted to reflect a preserving mechanism activated at reduced water temperatures. The changes in the haematological values in cod observed in the present study are probably an adaptive response caused by an increased oxygen demand with increasing water temperature, which is further enhanced by a reduced oxygen tension in the water. A reduced size of the erythrocytes implies a shorter mean diffusion path for oxygen, which, in addition to a higher concentration of erythrocytes, would make the oxygen uptake over the gills as well as the oxygen release at the different tissues more efficient (Moyle and Cech, 1982). Cameron (1970) found that changes in RBC and Hb in pinfish (Lugodon rhomboides) are of some significance in meeting seasonal variations in respiratory demands. Zanjani et al. (1969) assumed that Hb related to the oxygen requirement may act as a control mechanism in erythropoiesis in teleost fish. This was supported by HSirdig and Hoglund (1983); according to
140
these authors, fish blood contains a heterogeneous erythrocyte population in which the immature red cells are generally smaller and contain less haemoglobin than older mature ones. The present results may support this, as an increasing number of erythrocytes with reduced average size was found with increased water temperature. Table 2 shows the fatty acid composition of the erythrocyte lipids of the fish. The phosphatidylcholine (PC) was rich in 16:0 ( > 20% ) and the long-chain monoenes (20-24)) and the level of 20:5a3 was nearly as high as that of 22:6o3. The effect of temperature is reflected in the proportion of 20503 and 22:603 at 8’ C compared to 12 and 16’ C, and in a lower level of monoenoic fatty acids. Addison and Ackman (1971) reported similar values for the fatty acid composition of PC in adult cod erythrocytes. Selivonchick et al. (1977) reported similar effects in the fatty acid composition of PC from the brain of goldfish (Curassius auratus L. ). They showed that the increase in PUFA was due to TABLE 2 Fatty acid composition of erythrocyte phospholipids from cod fed for 9 weeks at different temperatures Temp. (“C)
Phosphatidylcholine
Phosphatidylserine
Phosphatidylethanolamine
Phosphatidylinositol
16
12
8
16
12
8
16
12
8
16
12
8
14:o 16:0 l&O IS
1.5 25.6 2.3 29.4
1.7 25.5 2.0 29.2
1.9 23.2 1.8 26.9
0.5 14.3 10.3 25.1
0.5 13.6 7.6 21.7
0.4 11.2 5.8 17.4
0.5 10.3 14.7 25.5
0.2 10.4 12.2 23.0
0.5 9.4 13.6 23.5
0.6 9.6 35.8 46.0
0.5 9.8 30.8 41.1
0.3 6.9 29.9 37.1
1611 18:lw9 18:lw7 2O:ll 22:l’ 24:l EM
2.0 13.2 2.0 5.3 4.8 3.6 30.9
2.1 12.8 2.0 5.5 3.9 2.6 28.9
2.1 9.6 1.9 5.4 3.4 2.3 24.7
0.8 6.2 4.2 10.2 2.6 24.0
0.7 6.9 4.6 10.7 2.1 25.1
0.8 6.1 4.4 10.8 1.7 23.8
1.0 4.2 1.8 6.9 9.2
1.2 4.6 1.7 6.6 6.8
0.7 3.6 2.1 8.1 6.4
0.9 4.8 2.0 5.1 1.5
1.1 6.8 2.5 5.3 1.1
0.8 5.6 2.4 5.0 1.0
23.1
20.9
20.9
14.3
16.8
14.8
18:20x 20:4w6 20:403 20~503 22~5~3 22:603 IP
1.5 0.9 0.4 16.0 0.7 18.7 38.2
1.6 0.8 0.2 15.6 0.7 20.6 39.5
1.7 0.9 0.4 17.8 0.9 24.4 46.1
1.2 1.0 0.3 11.9 0.9 32.1 47.4
1.7 1.5 0.3 10.4 1.0 36.2 51.1
1.7 1.3 0.4 13.2 1.1 38.6 56.3
0.6 0.4 0.1 4.8 0.8 41.0 47.7
0.8 0.5 0.2 8.7 0.7 40.8 51.7
0.7 0.4 0.1 3.9 0.7 45.9 51.7
0.7 6.1
1.2 6.3
0.8 9.0
24.2 0.3 6.1 37.4
24.8 0.4 7.1 39.8
30.0 0.5 7.1 47.4
Total 1
98.5
97.6
97.7
96.5
97.8
97.5
96.3
95.6
96.1
97.7
97.7
99.3
*Sum of isomers.
141
increases of Z&406 and 22:6m3 with a reduction mainly in 18:1m9. Cossins (1977) reported a similar general pattern for the fatty acid composition of PC from synaptosomal membrane preparations of goldfish adapted to 5 and 25 ‘C. An increase in polyunsaturated fatty acids (PUFA) (mainly 20:5 and 22:6) of PC of liver from rainbow trout acclimated to 5 and 20°C and a significant reduction in the level of the saturated fatty acids (mainly 16:0) were reported by Hazel (1979). The phosphatidylethanolamine (PE ) fraction of the erythrocytes had typically high levels of 22:6~3,20:1 and l&O, and a low level of 16:0 compared to PC. As for PC, the PUFA of the PE fraction were highest at the lowest temperature. But unlike PC, this temperature adaptation was not balanced by a simultaneous reduction in the level of monoenes, but of the saturated 16:0 and 18:0 fatty acids. This is generally consistent with the findings of Selivonchick et al. (1977) (PE from brain of goldfish), and Hazel (1979) (PE from liver of rainbow trout). The phosphatidylserine (PS ) fraction contained a high level of 22:6w3 and about equal levels of the long-chain monoenes with 20 and 22 carbons (6-9% ) . Of the saturated fatty acids, the l&O was higher than the 16:0. The fatty acid composition of this lipid fraction was not much affected by temperature during the growth period. This composition is different from that found by Addison and Ackman (1971)) but their values of PS fatty acids were based on one single analysis. Hazel (1979) observed an increase in the level of the PUFA in the liver PS of rainbow trout acclimated to 5 ’ C relative to 20 oC. The phosphatidyl inositol (PI) fraction is characterized by high levels of l&O, 20:4ti6 and 20:503. The level of polyunsaturated fatty acids (20~4~6 and 20:5w3) was higher in fish grown at 8°C than at 16”C, balanced by a lower level of the saturated fatty acids (160 and 18:O) at the lowest of these temperatures. Similar increases in the PUFA were observed in trout liver PI by Hazel (1979), due to 20:4w6 and 20:5w3, but in this tissue the arachidonic acid was the dominating fatty acid. The relatively high level of arachidonic acid is noteworthy and corresponding values have been observed in PI from other organs in cod (Bell et al., 1983; Tocher and Sargent, 1984; Lie and Lambertsen, 1987). As for the haematological values, the erythrocyte phospholipid fatty acid composition showed induced changes in response to a change in the environmental temperature. The four phospholipids examined all exhibited the same general pattern of fatty acid change in response to reduced water temperature. The levels of both the saturated and the monoene fatty acids declined while the sum of polyunsaturated fatty acids increased in the erythrocyte phospholipids. However, different specific fatty acids were responsible for the changes in the different phospholipid groups. According to Cossins (1977) the temperature-induced fatty acid changes in biological membranes should ensure a functional liquid-crystalline phase to
142
retain appropriate fluidity regardless of ambient temperature. He also pointed out that this acclimation can prevent excessive membrane mobility at higher temperature that may lead to heat injury and death in poikilotherms. How the biosynthetic processes are modulated to produce an adaptive shift in membrane fluidity is not yet clear. Thompson (1980) has suggested that vertical movements of biosynthetic enzymes in the membrane may induce variation in the order of the membrane in such a way that either the specificity or activity of fatty acid desaturases is modified. On the other hand, Melchior and Steim (1977) suggested that the differential solubility of saturated and unsaturated fatty acids in the membrane may provide the required fluidity programmed selectivity. However, according to Lands (1980) there is no evidence for a direct “dialogue” between membrane fluidity and biosynthesis. The relative proportions of the different phospholipids were not measured in this study, but Hazel (1979) found a relative accumulation of PE in the liver tissue of cold-acclimated trout which he suggested may represent an additional adaptation for increasing the degree of the membrane unsaturation, since PE is the most highly unsaturated phospholipid in trout liver. Selivonchick et al. (1977) pointed out the importance of plasmalogens, not separated from the diacyl phospholipids in this study, claiming that the maintenance of fluidity is achieved by changes both in plasmalogen content and in the unsaturation of the acyl and alkenyl chain. In a study with goldfish brain they demonstrated that in plasmalogen-rich myelin the changes relied mainly on the plasmalogen content. In the mitochondrial fraction, with a low plasmalogen content, the changes were mainly caused by the amounts of PUFA and the saturated fatty acids. Lee et al. (1986) measured the transition temperature between a gel phase and a liquid-crystalline phase in a mammalian membrane system and showed that a liquid-crystalline phase can be achieved without the use of PUFA. PC with l&O and 18:l in position 1 and 2, respectively, gave a transition temperature of 5’ C, and with 18:l in both positions the transition temperature was - 20°C. In addition to structural functions, one must also consider the metabolic role of fatty acids, which in certain phospholipids act as precursors for prostaglandins and leukotrienes. Greene and Selivonchick (1987) discussed the pathways taken by the PUFA in phospholipids, including incorporation into prostaglandins, and stated that temperature acclimation studies certainly indicate that this is not simply a matter of melting points and maintaining membrane fluidity. The changes observed in the haematological values with increasing temperature obviously increase the capacity for oxygen transport from the gills to the different tissues. However, the erythrocytes constitute a metabolizing tissue and the state of this metabolism can have important consequences in modifying the oxygen transport function of the haemoglobin. Fish erythrocyte me-
143
tabolism is not known in detail, but Riggs (1970) suggested that the overall functions are similar to those of red cells of other animals. According to Beutler ( 1986)) metabolic energy in human erythrocytes is needed to maintain the iron of haemoglobin in the divalent form, proper ionic environment (high K+, low Ca2+ and Na+ ), the sulphhydryl groups of enzymes and of haemoglobin in an active, reduced form, and to support the shape of the red cell. Membrane-bound enzymes in fish function in a similar manner to those in mammals (Bell et al., 1986 ), and according to Lee et al. (1986 ) ( Ca2+-Mg2+ ) ATPase activity is markedly dependent on the chemical structure of the surrounding phospholipids. Many membrane transport processes involving (Na+K+ ) -ATPase, Ca2+-ATPase and Mg ‘+-ATPase are sensitive to fatty acid composition of the membrane. Thomson et al. (1977) demonstrated that (Na+K+ ) -ATPase from eel gills is sensitive to changes in temperature, which again is correlated to the degree of unsaturation of the fatty acids. The data presented in the present paper support the theory that the PUFA have important metabolic functions in the erythrocyte in addition to their role in membrane functionality. These aspects may be of particular importance in poikilothermic animals like the cod, to secure optimal physiological function during changing environmental temperatures. ACKNOWLEDGEMENTS
The work was supported by a grant from the Norwegian Fisheries Research Council project. The skilled technical assistance of Miss Leikny Fjeldstad and Mrs Betty Irgens is gratefully acknowledged.
REFERENCES Addison, R.F. and Ackman, R.G., 1971. Erythrocyte lipids of Atlantic cod, Gadus morhua. Can. J. Biochem., 49: 873-876. Bell, M.V., Simpson, C.M.F. and Sargent, J.R., 1983. (n-3 ) and (n-6) polyunsaturated fatty acids in the phosphoglycerides of salt-secreting epithelia from two marine fish species. Lipids, 18: 720-726. Bell, M.V., Henderson, R.J. and Sargent, J.R., 1986. The role of polyunsaturated fatty acids in fish. Comp. Biochem. Physiol., 83: 711-719. Beutler, E., 1986. Energy metabolism and maintenance of erythrocytes. In: W.J. Williams, E. Beutler, A.J. Erslev and M.A. Lichtman (Editors), Hematology. McGraw-Hill Book Company, New York, NY, pp. 331-345. Cameron, J-N., 1970. The influence of environmental variables on the haematology of pinfish (Lagodon rhomboides) and striped mullet (Mugil cephalus). Comp. Biochem. Physiol., 32: 175-192.
Cossins, A.R., 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. Geurts van Kessel, W.S.M., Hax, W.M.A., Demel, R.A. and De Geir, J., 1977. High performance
144 liquid chromatographic separation and direct ultraviolet detection of phospholipids. Biochim. Biophys. Acta, 486: 524-530. Greene, D.H.A. and Selivonchick, D.P., 1987. Lipid metabolism in fish. Prog. Lipid Res., 26: 5385. H~dig, J. and Heglund, L.B., 1983. On accuracy in estimating fish bloodvariables. Comp. Biochem. Physiol., 75A: 35-40. Hazel, J.R., 1979. Influence of thermal acclimation on membrane lipid composition of rainbow trout. Am. J. Physiol., 236: R91-RlOl. Houston, A.H., 1980. Components of the hematological response of fishes to environmental temperature change. In: M.A. Ali (Editor), Environmental Physiology of Fishes. Plenum, New York, NY, pp. 241-298. Lands, W.E.M., 1980. Dialogue between membranes and their lipid-metabolising enzymes. Trans. Biochem. Sot., 8: 25-27. Lane, H.C., 1979. Progressive changes in haematology and tissue water of sexually mature trout, Salmo gairdneri Richardson, during the autumn and winter. J. Fish Biol., 15: 425-436. Lee, A.G., East, J.M. and Froud, R.J., 1986. Are essential fatty acids essential for membrane function? Prog. Lipid Res., 25: 41-46. Lie, 0. and Lambertsen, G., 1987. Deposition and distribution of fatty acids in tissue lipids in aquaria fed cod (Go&s morhaa). J. Am. Oil Chem. Sot., 64: 666. Lie, 0., Lied, E. and Lambertsen, G., 1986. Liver retention of fat and fatty acids in cod (Gadus morhua) fed different oils. Aquaculture, 59: 187-196. Melchior, D.L. and Steim, J.M., 1977. Control of fatty acid composition of Acholeplasma laidlawii membranes. Biochim. Biophys. Acta, 466: 148-159. Moyle, P.B. and Cech, J.J., 1982. Fishes: an Introduction to Ichthyology. Prentice-Hall, Englewood Cliffs, NJ, 593 pp. Munkittrick, K.R. and Leatherland, J.F., 1983. Haematocrit values in feral goldfish, Carassius auratus L., as indicators of the health of the population. J. Fish Biol., 23: 153-161. Riggs, A., 1970. Properties of fish hemoglobins. In: W.S. Hoar and D.J. Randall (Editors), Fish Physiology. Academic Press, New York, NY, pp. 209-252. Sandnes, K., Lie, 0. and Waagbe, R., 1988. Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon (Salmo salar). J. Fish Biol., 32: 129-136. Selivonchick, D.P., Johnston, P.V. and Roots, B.I., 1977. Acyl and alkenyl group composition of brain subcellular fraction of goldfish (Carassius auratus L.) acclimated to different environmental temperatures. Neurochem. Res., 2: 379-393. Thompson, G.A., 1980. Regulation of membrane fluidity during temperature acclimation by Tetrahymena pyriformis. In: M. Kates and A.A. Kuksis (Editors), Membrane Fluidity: Biophysical Techniques and Cellular Regulation. Humana Press, Clifton, NJ, pp. 381-397. Thomson, A.J., Sargent, J.R. and Owen, J.M., 1977. Influence of acclimatization temperature and salinity on (Na+ + K+ )-dependent adenosine triphosphatase and fatty acid composition in the gills of the eel (Anguilla anguilla). Comp. Biochem. Physiol., 58B: 223-228. Tocher, D.R. and Sargent, J.R., 1984. Analyses of lipids and fatty acids in ripe roes of some northwest European marine fish. Lipids, 19: 492-499. Zanjani, E.D., Yu, M.L., Perlmutter, A. and Gordon, A.S., 1969. Humoral factors influencing erythropoiesis in the fish blue gourami (Trichogaster trichopterus). Blood, 33: 573-581.
137 in The Netherlands
Haematological Values and Fatty Acid Composition of Erythrocyte Phospholipids in Cod (Gadus morhua) Fed at Different Water Temperatures 0YVIND LIE, EINAR LIED and GEORG LAMBERTSEN Institute of Nutrition, Directorate of Fisheries, P.0. Box 1900, N-5024 Bergen (Norway)
ABSTRACT Lie, O., Lied, E. and Lambertsen, G., 1989. Haematological values and fatty acid composition of erythrocyte phospholipids in cod (Godus morhua) fed at different water temperatures. Aquaculture, 79: 137-144. Haematological values and fatty acid composition of erythrocyte phospholipids were determined in cod (Gadus morhua) which had been fed at 812 and 16” C. The number of erythrocytes and the values of haematocrit and haemoglobin were highest at 16 OC whereas the mean cell volume and mean cell haemoglobin were lowest at this temperature. The mean cell haemoglobin concentration was not affected by water temperature. The fatty acid composition of the main phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE ) , phosphatidylserine (PS) and phosphatidylinositol (PI), in the erythrocytes was determined. The relative levels of both saturated and monoene fatty acids declined with decreasing water temperature and the polyunsaturated fatty acid fraction generally increased.
INTRODUCTION
Fish haematological values undergo seasonal variations concomitant to climatic changes in light and water temperature (H&dig and Hoglund, 1983). The effects of temperature on lipid composition of fish have recently been reviewed by Bell et al. (1986) and Greene and Selivonchick (1987)) showing that the ratio of polyunsaturated to saturated fatty acids in fish membrane lipids increases in response to lowered ambient water temperature. Erythrocyte metabolism and function are dependent on the functionality of the membrane, wherein many of the important metabolic and transport systems of the cells are embedded. We have earlier demonstrated that the fatty acid composition of fish lipids is affected by the fatty acids present in the feed lipid. In the present paper we describe how the fish is able to adjust its lipid composition according to temperature.
0044-8486/89/$03.50
0 1989 Elsevier Science Publishers B.V.
138 MATERIALS AND METHODS
Fish and diets. Cod (mean weight 180 g) were obtained from culture at the Aquaculture Station Austevoll (Institute of Marine Research, Directorate of Fisheries, Norway). Three groups each of 20 fish were kept in aquaria of 175 1 for 9 weeks at 8,12 and 16°C after a gradual temperature acclimatization for 4 weeks. The fish were fed to satiation once a day 5 days a week. The diet was based on squid mantle (91.4% ), capelin oil (4.1% ), dextrinized potato starch (4.3% ) and a mixture (0.2% ) of vitamins and minerals according to Lie et al. (1986). Analytical methods. Blood samples were collected from the ductus cuvieri and treated according to Sandnes et al. (1988). Red blood cell count (RBC ), haematocrit (Hct ) and haemoglobin (Hb ) were determined according to Sandnes et al. (1988). A portion of the blood was centrifuged at 3000 g and the erythrocytes were washed three times in an isotonic saline solution. Total lipid was extracted from washed erythrocytes with chloroform/methanol (2:1, u/u). The four phospholipid groups phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS) and phosphatidylinositol (PI ) were separated by an adsorption HPLC method modified from Geurts van Kessel et al. (1977) (Lie and Lambertsen, to be published). Each phospholipid fraction was saponified and the fatty acids were methylated in 12% borontrifluoride in methanol. The methyl esters were analysed using a Carlo Erba 2900 gas chromatograph equipped with a 50-m CP-sil88 (Chrompack) fused silica capillary column (i.d. 0.32 mm). The fatty acid composition was calculated using an integrator (Shimadzu C-R3A) and peak identification was ascertained by a standard mixture of methyl esters (Nu-Chek Prep., Elysian, MN, U.S.A.). Statistics. The analytical data were evaluated statistically by means of the Student’s t-test. RESULTS AND DISCUSSION
The analytical data presented in this paper relate to blood samples from three groups of cod which were allowed to grow for 9 weeks at 8,12 and 16°C on a moist diet composed of squid mantle and capelin as the protein and lipid source. During this period the cod grew from an average weight of 180 g to about 250 g at 8°C and to about 280 g at 12 and 16°C. Table 1 shows that the fish was able to adjust its blood according to temperature during the growth period; i.e. the number of red blood cells (RBC), the haematocrit (Hct ) and haemoglobin (Hb) were significantly lower at 8” C than at 12 and 16 oC. The mean cell haemoglobin concentration (MCHC ) , however, was not affected by the temperature the fish had been subjected to, whereas
139 TABLE 1 Haematological values (mean k SD, n = 8) in cod held at different water temperatures Significant differences between groups
Temperature ( “C)
16 RBC (~10~~ 1-l) SD Hct (%) SD Hb (g 100 ml-l) SD MCH (x10-6g-‘) SD MCV (x lo-l5 1-l) SD MCHC (g 100 ml-‘) SD
1.31 0.12 28.5 2.4 5.35 0.48 41 4 219 16 18.8 0.7
12
8
16-8
16-12
12-8
1.10
1.03
***
**
0.14 27.9
0.17 25.3
**
-
*
2.7 5.25 0.66 48
2.8 4.80 0.62 47
* **
***
_
**
***
3 256 22 18.8 0.8
6 249 31 19.0 0.9
-
*O.Ol
the mean cell haemoglobin (MCH) an the mean cell volume (MCV) were markedly lower at 16°C than at 8 and 12°C. Adaptation to elevated water temperature has been shown to induce a number of haematological changes (Houston, 1980). Lane (1979) reported a reduction in RBC, Hct and Hb in rainbow trout (SC&O guirdneri) during the period from October to March, but these parameters were not significantly correlated to water temperature. Similarly, Munkittrick and Leatherland (1983) reported a significant increase in Hct in goldfish (Carassius aurutus L. ) with increased water temperature. An increase in MCH and MCHC was reported by Lane (1979) in rainbow trout, and was interpreted to reflect a preserving mechanism activated at reduced water temperatures. The changes in the haematological values in cod observed in the present study are probably an adaptive response caused by an increased oxygen demand with increasing water temperature, which is further enhanced by a reduced oxygen tension in the water. A reduced size of the erythrocytes implies a shorter mean diffusion path for oxygen, which, in addition to a higher concentration of erythrocytes, would make the oxygen uptake over the gills as well as the oxygen release at the different tissues more efficient (Moyle and Cech, 1982). Cameron (1970) found that changes in RBC and Hb in pinfish (Lugodon rhomboides) are of some significance in meeting seasonal variations in respiratory demands. Zanjani et al. (1969) assumed that Hb related to the oxygen requirement may act as a control mechanism in erythropoiesis in teleost fish. This was supported by HSirdig and Hoglund (1983); according to
140
these authors, fish blood contains a heterogeneous erythrocyte population in which the immature red cells are generally smaller and contain less haemoglobin than older mature ones. The present results may support this, as an increasing number of erythrocytes with reduced average size was found with increased water temperature. Table 2 shows the fatty acid composition of the erythrocyte lipids of the fish. The phosphatidylcholine (PC) was rich in 16:0 ( > 20% ) and the long-chain monoenes (20-24)) and the level of 20:5a3 was nearly as high as that of 22:6o3. The effect of temperature is reflected in the proportion of 20503 and 22:603 at 8’ C compared to 12 and 16’ C, and in a lower level of monoenoic fatty acids. Addison and Ackman (1971) reported similar values for the fatty acid composition of PC in adult cod erythrocytes. Selivonchick et al. (1977) reported similar effects in the fatty acid composition of PC from the brain of goldfish (Curassius auratus L. ). They showed that the increase in PUFA was due to TABLE 2 Fatty acid composition of erythrocyte phospholipids from cod fed for 9 weeks at different temperatures Temp. (“C)
Phosphatidylcholine
Phosphatidylserine
Phosphatidylethanolamine
Phosphatidylinositol
16
12
8
16
12
8
16
12
8
16
12
8
14:o 16:0 l&O IS
1.5 25.6 2.3 29.4
1.7 25.5 2.0 29.2
1.9 23.2 1.8 26.9
0.5 14.3 10.3 25.1
0.5 13.6 7.6 21.7
0.4 11.2 5.8 17.4
0.5 10.3 14.7 25.5
0.2 10.4 12.2 23.0
0.5 9.4 13.6 23.5
0.6 9.6 35.8 46.0
0.5 9.8 30.8 41.1
0.3 6.9 29.9 37.1
1611 18:lw9 18:lw7 2O:ll 22:l’ 24:l EM
2.0 13.2 2.0 5.3 4.8 3.6 30.9
2.1 12.8 2.0 5.5 3.9 2.6 28.9
2.1 9.6 1.9 5.4 3.4 2.3 24.7
0.8 6.2 4.2 10.2 2.6 24.0
0.7 6.9 4.6 10.7 2.1 25.1
0.8 6.1 4.4 10.8 1.7 23.8
1.0 4.2 1.8 6.9 9.2
1.2 4.6 1.7 6.6 6.8
0.7 3.6 2.1 8.1 6.4
0.9 4.8 2.0 5.1 1.5
1.1 6.8 2.5 5.3 1.1
0.8 5.6 2.4 5.0 1.0
23.1
20.9
20.9
14.3
16.8
14.8
18:20x 20:4w6 20:403 20~503 22~5~3 22:603 IP
1.5 0.9 0.4 16.0 0.7 18.7 38.2
1.6 0.8 0.2 15.6 0.7 20.6 39.5
1.7 0.9 0.4 17.8 0.9 24.4 46.1
1.2 1.0 0.3 11.9 0.9 32.1 47.4
1.7 1.5 0.3 10.4 1.0 36.2 51.1
1.7 1.3 0.4 13.2 1.1 38.6 56.3
0.6 0.4 0.1 4.8 0.8 41.0 47.7
0.8 0.5 0.2 8.7 0.7 40.8 51.7
0.7 0.4 0.1 3.9 0.7 45.9 51.7
0.7 6.1
1.2 6.3
0.8 9.0
24.2 0.3 6.1 37.4
24.8 0.4 7.1 39.8
30.0 0.5 7.1 47.4
Total 1
98.5
97.6
97.7
96.5
97.8
97.5
96.3
95.6
96.1
97.7
97.7
99.3
*Sum of isomers.
141
increases of Z&406 and 22:6m3 with a reduction mainly in 18:1m9. Cossins (1977) reported a similar general pattern for the fatty acid composition of PC from synaptosomal membrane preparations of goldfish adapted to 5 and 25 ‘C. An increase in polyunsaturated fatty acids (PUFA) (mainly 20:5 and 22:6) of PC of liver from rainbow trout acclimated to 5 and 20°C and a significant reduction in the level of the saturated fatty acids (mainly 16:0) were reported by Hazel (1979). The phosphatidylethanolamine (PE ) fraction of the erythrocytes had typically high levels of 22:6~3,20:1 and l&O, and a low level of 16:0 compared to PC. As for PC, the PUFA of the PE fraction were highest at the lowest temperature. But unlike PC, this temperature adaptation was not balanced by a simultaneous reduction in the level of monoenes, but of the saturated 16:0 and 18:0 fatty acids. This is generally consistent with the findings of Selivonchick et al. (1977) (PE from brain of goldfish), and Hazel (1979) (PE from liver of rainbow trout). The phosphatidylserine (PS ) fraction contained a high level of 22:6w3 and about equal levels of the long-chain monoenes with 20 and 22 carbons (6-9% ) . Of the saturated fatty acids, the l&O was higher than the 16:0. The fatty acid composition of this lipid fraction was not much affected by temperature during the growth period. This composition is different from that found by Addison and Ackman (1971)) but their values of PS fatty acids were based on one single analysis. Hazel (1979) observed an increase in the level of the PUFA in the liver PS of rainbow trout acclimated to 5 ’ C relative to 20 oC. The phosphatidyl inositol (PI) fraction is characterized by high levels of l&O, 20:4ti6 and 20:503. The level of polyunsaturated fatty acids (20~4~6 and 20:5w3) was higher in fish grown at 8°C than at 16”C, balanced by a lower level of the saturated fatty acids (160 and 18:O) at the lowest of these temperatures. Similar increases in the PUFA were observed in trout liver PI by Hazel (1979), due to 20:4w6 and 20:5w3, but in this tissue the arachidonic acid was the dominating fatty acid. The relatively high level of arachidonic acid is noteworthy and corresponding values have been observed in PI from other organs in cod (Bell et al., 1983; Tocher and Sargent, 1984; Lie and Lambertsen, 1987). As for the haematological values, the erythrocyte phospholipid fatty acid composition showed induced changes in response to a change in the environmental temperature. The four phospholipids examined all exhibited the same general pattern of fatty acid change in response to reduced water temperature. The levels of both the saturated and the monoene fatty acids declined while the sum of polyunsaturated fatty acids increased in the erythrocyte phospholipids. However, different specific fatty acids were responsible for the changes in the different phospholipid groups. According to Cossins (1977) the temperature-induced fatty acid changes in biological membranes should ensure a functional liquid-crystalline phase to
142
retain appropriate fluidity regardless of ambient temperature. He also pointed out that this acclimation can prevent excessive membrane mobility at higher temperature that may lead to heat injury and death in poikilotherms. How the biosynthetic processes are modulated to produce an adaptive shift in membrane fluidity is not yet clear. Thompson (1980) has suggested that vertical movements of biosynthetic enzymes in the membrane may induce variation in the order of the membrane in such a way that either the specificity or activity of fatty acid desaturases is modified. On the other hand, Melchior and Steim (1977) suggested that the differential solubility of saturated and unsaturated fatty acids in the membrane may provide the required fluidity programmed selectivity. However, according to Lands (1980) there is no evidence for a direct “dialogue” between membrane fluidity and biosynthesis. The relative proportions of the different phospholipids were not measured in this study, but Hazel (1979) found a relative accumulation of PE in the liver tissue of cold-acclimated trout which he suggested may represent an additional adaptation for increasing the degree of the membrane unsaturation, since PE is the most highly unsaturated phospholipid in trout liver. Selivonchick et al. (1977) pointed out the importance of plasmalogens, not separated from the diacyl phospholipids in this study, claiming that the maintenance of fluidity is achieved by changes both in plasmalogen content and in the unsaturation of the acyl and alkenyl chain. In a study with goldfish brain they demonstrated that in plasmalogen-rich myelin the changes relied mainly on the plasmalogen content. In the mitochondrial fraction, with a low plasmalogen content, the changes were mainly caused by the amounts of PUFA and the saturated fatty acids. Lee et al. (1986) measured the transition temperature between a gel phase and a liquid-crystalline phase in a mammalian membrane system and showed that a liquid-crystalline phase can be achieved without the use of PUFA. PC with l&O and 18:l in position 1 and 2, respectively, gave a transition temperature of 5’ C, and with 18:l in both positions the transition temperature was - 20°C. In addition to structural functions, one must also consider the metabolic role of fatty acids, which in certain phospholipids act as precursors for prostaglandins and leukotrienes. Greene and Selivonchick (1987) discussed the pathways taken by the PUFA in phospholipids, including incorporation into prostaglandins, and stated that temperature acclimation studies certainly indicate that this is not simply a matter of melting points and maintaining membrane fluidity. The changes observed in the haematological values with increasing temperature obviously increase the capacity for oxygen transport from the gills to the different tissues. However, the erythrocytes constitute a metabolizing tissue and the state of this metabolism can have important consequences in modifying the oxygen transport function of the haemoglobin. Fish erythrocyte me-
143
tabolism is not known in detail, but Riggs (1970) suggested that the overall functions are similar to those of red cells of other animals. According to Beutler ( 1986)) metabolic energy in human erythrocytes is needed to maintain the iron of haemoglobin in the divalent form, proper ionic environment (high K+, low Ca2+ and Na+ ), the sulphhydryl groups of enzymes and of haemoglobin in an active, reduced form, and to support the shape of the red cell. Membrane-bound enzymes in fish function in a similar manner to those in mammals (Bell et al., 1986 ), and according to Lee et al. (1986 ) ( Ca2+-Mg2+ ) ATPase activity is markedly dependent on the chemical structure of the surrounding phospholipids. Many membrane transport processes involving (Na+K+ ) -ATPase, Ca2+-ATPase and Mg ‘+-ATPase are sensitive to fatty acid composition of the membrane. Thomson et al. (1977) demonstrated that (Na+K+ ) -ATPase from eel gills is sensitive to changes in temperature, which again is correlated to the degree of unsaturation of the fatty acids. The data presented in the present paper support the theory that the PUFA have important metabolic functions in the erythrocyte in addition to their role in membrane functionality. These aspects may be of particular importance in poikilothermic animals like the cod, to secure optimal physiological function during changing environmental temperatures. ACKNOWLEDGEMENTS
The work was supported by a grant from the Norwegian Fisheries Research Council project. The skilled technical assistance of Miss Leikny Fjeldstad and Mrs Betty Irgens is gratefully acknowledged.
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