Fish Physiology and Biochemistry vol. 5 no. 4 pp 219-227 (1988) Ktlgler Publications, Amsterdam/Berkeley
The fatty acid compositions of established fish cell lines after long-term culture in mammalian sera D.R. Tocher ~, J.R. Sargent I and G.N. Frerichs 2 I N E R C Unit o f Aquatic Biochemistry, Dept o f Biological Science, Universitv q f Stirling, Stirling FK9 4LA, Scotland, U.K. 2 Institute o f Aquaculture, University o f Stirling, Stirling FK9 4LA, Scotland, U.K. Keywords: fatty acids, compositions, fish cells, culture
Abstract The effect of long-term culture of fish cells in mammalian serum on the phospholipid fatty acid composition was investigated. All the cell lines studied had much lower levels of polyunsaturated fatty acids (PUFA) than those found in intact fish tissues. In particular (n-3)PUFA were considerably depleted in the cultured cell lines, leading to very low (n-3)/(n-6) ratios in all the phospholipid classes. In general the cells were rich in 18:1, 16:0, 18:0 and 16:1 with 20:4(n-6) and 22:6(n-3) as the major PUFA. The fatty acid composition reflected the composition o f the fetal calf serum added to the media rather than their fish tissue origins. The results were discussed in relation to the roles of PUFA in general cell metabolism and more specifically the role of (n-3)PUFA in fish cells.
Abbreviations PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PUFA, polyunsaturated fatty acids.
Introduction Whereas the PUFA of terrestrial mammals, including man, are dominated by the (n-6) series, in aquatic and especially marine organisms, (n-3) PUFA predominate (Sargent 1976; Sargent and Whittle 1981; Sargent et al. 1987). This is perhaps reflected in the qualitatively different essential fatty acid requirements for mammals and fish. Mammals have an absolute requirement for 18:2(n-6) (Burr and Burr 1930), whereas their requirement for 18:3(n-3) has been difficult to establish (Tinoco 1982). In contrast, fish have a well-defined require-
merit for 18:3(n-3) (Watanabe 1982; Greene and Selivonchick 1987) and also long chain (n-3)PUFA in the case of some marine fish (Watanabe 1982; Greene and Selivonchick 1987; Sargent et al. 1987) whereas the qualitative and quantitative requirements for 18:2(n-6) or other (n-6)PUFA have not been conclusively established (Watanabe 1982; Bell' et al. 1985; Sargent et al. 1987; Henderson and Tocher, 1987). It has been reported, however, that the majority of mammalian cell lines that have been investigated appear to grow well in the absence of PUFA, both essential and non-essential (Bailey and Dunbar 1973). However the authors of that study also observed that significant amounts of linoleic acid were consistently found in samples of commercially obtained chemically defined 'lipid-free' culture media (Bailey et al. 1972). It is routine for most tissue culture media to contain no lipid or essential fatty acids other than those present in the serum supple-
220 ment. The lipids and fatty acids thus available to tissue culture cells are dependent upon the type of serum utilized in the medium. Fish cell culture was fi'rst established almost 30 years ago and, as with mammalian cells, serum was found to be an essential supplement (Wolf and Quimby 1969). Sera from many species, including homologous and heterologous fish, have proved very variable in growth promoting properties (Wolf and Quimby 1969). Furthermore, the considerable difficull, ies in obtaining sufficient fish serum for cell culture combined with the risk of introducing latent fish viruses has determined that fetal calf serum has been the serum of choice for fish cell culture as with mammalian cell culture. In this study we have determined the fatty acid profile of a variety of teleost fish cell lines to investigate the effect that long-term culture in mammalian serum has had on PUFA levels, and particularly the (n-3)PUFA content. In this report we compare and discuss the fatty acid compositions of the different phospholipid classes in the various fish cell lines.
Materials and methods
Cell lines Rainbow trout (Sahno gairdneri) gonad (RTG-2) (Wolf and Quimby 1962), bluegill (Lepomis macrochirus) fry (BF-2) (Wolf et al. 1966), fathead minnow (Pimephales promelas) (FHM) (Gravell and Malsberger 1965) and Atlantic salmon (Salmo salar) (AS) (Nicholson and Byrne 1973) cell lines were obtained originally from Flow Laboratories, Rickmansworth, U.K. The chinook salmon (Oncorhynchus tshawytscha) epithelium cell line (CHSE-214) was obtained from the Microbiology section, Institute of Aquaculture, University of Stifling. All these cell lines had been maintained in continuous culture in the Institute of Aquaculture for many passages. The turbot (Scophthalmus maximus) fin cell line (TF) was a gift from Dr. B. Hill, MAFF Fish Diseases Laboratory, Weymouth, U.K., who had originally established the line.
Media and growth conditions AS, BF-2 and CHSE-214 cells were grown in minimum essential medium Eagle (modified) with Earle's salts containing 0.25~ bicarbonate and 1% non-essential amino acids. RTG-2 cells were grown in Glasgow's modification of Eagle's medium containing 16 mM Tris-HC1 buffer (Trizma, pH 7.4), 0.3~ sodium bicarbonate and 10~ tryptose phosphate broth. Both FHM and TF cells were grown in Leibovitz L-15 medium, with 0.35~ sodium chloride added in the case of TF cells. All media were further supplemented with 10070 fetal calf serum and antibiotics (50 I.U./ml penicillin and 50 ~g/ml streptomycin). The cell cultures were grown to confluence in sealed 75 cm: plastic flasks (Gibco Ltd., Paisley, U.K.) at 22~
Cell harvesting and lipid extraction Cells were harvested within 24 h of reaching confluence. Media were decanted from the flasks and the monolayers washed with phosphate buffered saline before addition of 1 ml Trypsin-EDTA (0.05~ and 0.02~ respectively). When the cells were dissociated from the substrate, 2 ml of the appropriate medium were added to inhibit further action of the trypsin. The cells were centrifuged at 600 • g for 10 mins at 4~ the supernatant poured off and the cell pellet washed 3 times with 10 ml phosphate buffered saline to ensure removal of all serum. Total lipid was extracted from the washed cells essentially according to Folch et al. (1957) by addition of 5 ml ice cold c h l o r o f o r m / m e t h a n o l (2:1, v/v) containing 0.05~ butylated hydroxytoluene. After homogenisation in a glass-teflon pestle homogeniser, the extract was filtered and 1 ml 0.88o7o KCI added. Following mixing and separation of the phases by centrifugation, the chloroform layer was taken and dried down under nitrogen before being redissolved in c h l o r o f o r m / methanol (2:1, v/v) containing 0.05~ butylated hydroxytoluene at a final lipid concentration of I00 mg/ml.
221
Lipid and .fatty acid analysis Lipid class analysis was performed by thin-layer chromatography-flame ionisation detection (TLCFID) using an latroscan TH-10 mk.lV (latron Laboratories, Tokyo) equipped with a HewlettPackard HP3300 recording integrator. Precise details of methodology and calibration of the instrument have been described previously (Fraser et al. 1985). Total lipid was separated into individual neutral and phospholipid classes by l-dimensional and 2-dimensional TLC, respectively, as described previously (Tocher and Sargent 1984). Fatty acid methyl esters were prepared by acid-catalysed transmethylation (Christie 1982) with subsequent purification of the extracted methyl esters by TLC. The methyl esters were analysed on a Packard 436 gas chromatograph (Packard Instruments Inc., Caversham, U.K.) equipped with a chemically bonded CP Wax 52CB fused silica capillary column (50 m • 0.34 mm i.d.) (Chrompack U.K. Ltd., London), an on-column injector and using H, as carrier gas. Details of operating conditions and identification of fatty acid methyl esters were as described previously (Tocher and Sargent 1984; Tocher et al. 1986). Data are means of triplicate GC analyses; standard deviations are omitted for clarity but were generally less than 507o.
Materials All media, phosphate buffered saline, sodium bicarbonate, non-essential amino acids, tryptose phosphate broth, antibiotics, fetal call" serum and trypsin-EDTA were obtained from Flow Laboratories, Rickmansworth, U.K. Butylated hydroxytoluene and Trizma were from Sigma Chemical Co. Ltd., Poole, U.K. All solvents were H P L C grade and were obtained from Rathburn Chemicals, Walkerburn, U.K.
Results
All the cell lines used in the present study were known to have been maintained in culture media
containing mammalian sera, almost exclusively fetal calf serum, for many passages prior to the onset of the experiment, and for 3 passages during the experiment before harvesting. Phospholipids accounted for from over 72% to under 41% of the total lipid, decreasing in the order RTG-2 > TF > AS > FHM > BF-2 > CHSE-214 (Table 1). Triacylglycerol content was inversely related to the total phospholipid and increased in the cells in the same order as above, rising from 2.2% in RTG-2 cells to 28.4% in CHSE-214. Sterol esters varied and did not relate to the neutral lipid or triacylglycerol levels. Cholesterol was more constant averaging approximately 19% in most cell lines other than FHM which had 12.4%. Phosphatidylcholine (PC) was the major phospholipid class in all cases but was highly variable ranging from 19.4% to 40.8% of total lipid, whereas phosphatidylethanolamine (PE) was more constant at around 20% (Table 1). RTG-2 cells were notable for their high content (11.1~ of phosphatidylserine (PS)/phosphatidylinositol (PI) in comparison with the other cells. Fetal calf serum lipid was primarily sterol ester and triacylglycerol with lesser amounts of PC, sphingomyelin and free cholesterol. In total lipid, monoenes (primarily 18:1(n-9)) accounted for approximately 40%-60~ of the total fatty acids with saturates (primarily 16:0 and 18:0) accounting for between 27% and 36% (Table 2). Total PUFA was highest in RTG-2 and TF at almost 25~ and 24% respectively but was only 9% in AS and BF-2. The (n-6)PUFA predominated over (n-3)PUFA by 2-fold (RTG-2) to over 3-fold ' (CHSE-214). Arachidonic acid (20:4).and 22:6 were the major (n-6) and (n-3)PUFA respectively. Of the individual phospholipid classes, PC was lowest in PUFA, having levels lower than those in total lipid ranging from 8.2% to 12.0070 (Table 3) with CI8 PUFA more prominent as compared with total lipid. Monoenes were slightly lower in PC than in total lipid but saturates were considerably higher, due to increased 16:0. The highest levels of PUFA were found in PE and P S / P i in all cell lines (Tables 4 and 5). In PE and P S / P I , total PUFA ranged from 21.9 to 36.2% and from 10.5 to 23.7%, respectively. In general 18: l(n-9), 16:0, 18:0, 16:1,
222
Table I. Lipid class composition of the cell lines and fetal calf serum (FCS) Cell
line
Lipid
RTG-2
TF
AS
CHSE-214
% Phospholipids
72.4
65.8
61.7
40.5
46.8
56,1
13,2
% Neutral lipids
27.6
34.2
38,3
59.5
53.2
43.9
86.8
36.7 21.5
35.4 23.7
40.8 19.4
19.4 17.3
24.7 19.7
31. l 21,1
8.2 -
I I. I 3. I
3.5 3. I
0,4 I. I
1,0 2.8
0.8 1,6
1.4 2.5
4.8
2.2 2.2 1.8 21.3
9.9 4.6 2.6 17.2
3.2 13.9 2.3 18.9
9.7 28.4 2.0 19.3
5,6 24.7 3.7 19.0
8.4 21.3 1.7 12.4
58. I 24.2 0.8 3.7
Phospholipid classes: (% total lipid) Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine/ inositol Sphingomyelin Neutral classes: total lipid) Sterol ester Triacylglycerol Free fatty acid Cholesterol
BF-2
FHM
FCS
(%
-
= not detected.
Table 2. Fatty acid compositions of total lipid from cell lines and fetal calf serum Cell line Fatty acid
RTG-2
TF
AS
CHSE-214
BF-2
FHM
FCS r
14:0 16:0 18:0 Total saturates a
1.2 13.8 9.6 27.0
2.2 17.7 12.8 36.4
1.7 16.1 6.6 26.7
1.4 15.0 7.3 26.6
1.1 18.6 11.7 35.6
1.2 15.8 9.7 29.4
2.6 22.1 11.1 39.3
16:1 b, 18:1(n-9) 18:1(n-7) Total monoenes c
8.7 32.6 4.5 47.9
10.1 24.3 3.9 38.9
9.6 45.7 3.8 61.0
10.5 25.5 5.4 50.4
15.3 21.9 4.0 43.9
9.8 33.7 5.7 52.2
5.2 18.7 5.6 31.9
18:2(n-6) 18:3(n-6) 20:2(n-6) 20:3(n-6) 20:4(n-6) Total (n-6)PUFA d
1.8 1.4 1.8 1.6 8.4 16. I
2.5 0.5 0.2 1.5 8.6 15.9
1.5 0.3 0.2 0.6 3.4 6.3
1.8 0.4 0.7 1.4 5.2 9.9
0.8 0.6 0.6 0.2 4.0 6.4
1.2 0.1 2.0 1.5 4.8 9.9
4.7 0.2 0.3 1.9 8.9 16.6
-
-
-
0.3
0.5
0.5
0.3
0.2
-
-
2.3 3.4 6.4
2.3 5.2 7.8
0.9 1.1 2.2
1.0 2.0 3.0
18:2(n-9)
-
1.0
.
20:3(n-9)
2.4
-
0.5
1.8
24.9 0.40
24.7 0.49
9.0 0.35
14.7 0.30
18:3(n:.3) 20:5{n-3) 22:5(n-3) 22:6(n-3) Total (n-3)PUFA c
Total PUFA (n-3)/(n-6)
0.2
.
-
0.3 1.8 2. I .
0.6
1.1 2.0 3.4
2.2 3.3 6.6
0.5
3.4
0.2
9.0 0.33
16.7 0.34
24.0 0.40
.
.
Results are expressed as a % of weight: aalso includes 15:0, 17:0, 20:0 and 22:0; bpredominantly (n-7) isomer: ':also includes 20:1 isomers, 22: I and 24. I : dalso includes 22:4(n-6); Calso includes 18:4(n-3) and 20:4(n-3); raverage of the two batches of FCS used in this study; - = not detected.
223
Table 3. Fatty acid composition of phosphatidylcholine from cell lines Cell line Fatty acid
RTG-2
TF
AS
CHSE-214
BF-2
FHM
14:0 16:0 18:0 Total saturates ~'
5.2 28.2 9.8 52.6
4.5 26.2 7.3 43.9
3.0 21.8 5.8 35.3
1.8 23.2 7.6 39.1
3.0 25.7 7.3 43.3
2.7 23.7 8.0 40.7
16:1 b 18: I (n-9) 18: I (n-7) Total monoenes c
13.4 19.5 2.1 36.0
14.3 23.8 3.2 42.6
13.0 38.9 3.5 56.1
10.8 31. I 4.4 48.6
20.0 24.0 3.0 48.3
13.7 32.0 3.8 50.5
18:2(n-6) 18:3(n-6) 20:3(n-6) 20:4(n-6) Total (n-6)PUFA a
1.8 2.8 1.4 6.0
2.3 1.3 0.9 3.2 8.1
2. I 1.2 0.2 0.7 4.2
3.0 1.9 1.4 1.4 7.7
2.5 0.9 0.3 0.8 4.5
2.1 1.0 0.6 1.0 4.7
22:501-3) 22:6(n-3) Total (n-3)PUFA ~'
O. I 3.4 3.5
0.5 2.9 3.4
0.2 I. I 4.0
0.1 1.6 3.3
0. I 2.0 3.4
1.8 2.4
0.2
. 1.0
0.3
1.6
8.4 0.95
12.0 0.43
8.2 0.78
8.7 0.51
18:2(n-9) 20:3(n-9)
.
Total PUFA (n-3)/(n-6)
9.5 0.58
I 1.5 0.42
.
.
Notes as described in Table 2 except ':also includes 18:3(n-3), 18:4(n-3), 20:4(n-3) and 20:5(n-3).
Table 4. Fatty acid composition of phosphatidylethanolamine from cell lines Cell line Fatty acid
RTG-2
TF
AS
CHSE-214
BF-2
FHM
14:0 16:0 18:0 Total saturates a
4.9 13.8 10.4 36.7
1.9 12.4 13.4 32.2
1.9 13.2 5.6 26.5
1.0 12.0 7.6 26.0
2.9 16.0 I 1.8 36.5
1.7 1I.I 8.8 26.9
16:1 t' 18: l(n-9) 18:1(n-7) Total monoenes c
9.7 19.6 1.5 33.3
7.7 16. I 3.9 28.5
12.3 34.7 2.6 50.8
7.6 23.8 5.9 45.4
13.9 17. I 4.2 37.9
8.4 27.0 " 5.2 43.8
18:2(n-6) 18:3(n-6) 20:2(n-6) 20:3(n-6) 20:4(n-6) Total (n-6)PUFA d
3.0 1.3 I.I 0.6 10.3 16.5
2.7 1.8 0.5 1.4 14.4 23.6
3.8 1.9 0.4 0.9 5.1 12.4
3.5 2.1 0.5 1.8 6.7 15.6
2.7 3.5 0.8 0.8 3.9 12.9
2.7 I.I 3.6 1.8 7.4 17.2
22:5(n-3) 22:6(n-3) Total (n-3)PU FA c
2.0 5.5 7.5
3.0 7.4 12.0
1.5 3.9 7.6
1.8 4.6 9.6
1.8 4.7 9.2
1.4 3.8 7.0
18:2(n-9) 20:3(n-9)
1.9
0.6 -
. 1.9
3. I
1.5
4.8
Total PUFA (n-3)/(n-6)
25.9 0.46
36.2 0.51
21.9 0.61
28.3 0.62
23.6 0.71
29.0 0.41
Notes as described in Table 3.
.
.
.
224 Table 5. Fatty acid composition of phosph/uidylserirle/phosphatidylinositol from cell lines Cell line Fatty acid
RTG-2
TF
AS
CHSE-214
BF-2
FHM
14:0 16:0 18:0 Total saturales a
5.3 25.2 13.2 51.8
3.6 20.7 19.9 50.6
1.5 16.5 13.3 38.0
6.8 19.1 I 1.9 45.8
2.4 19.6 17.3 49.0
3.8 20.7 14.1 45.0
16:1 b 18:1(n-9) 18:11n-7) Total mofioencs c
I 1.4 13.5 1.8 28.3
8.9 13.8 1.4 25.6
9.2 24.5 2.2 39.0
9.6 12.1 1.9 25.3
11.8 14.9 I.I 29.0
2.3 16+1 1.7 32.3
18:2(n-6)
2.1
1.4
2.5
2.9
3.1
2.6
18:31n-61 20:2(n-61 20:31n-6)
1.8 0.6
1.3 0.2 1.2
2.1 .0.8 1.8
3.9 0.8 1.8
3.4 0.6 1.0
1.3 2.8 1.3
20:41n-61 Total (n-6)PU FA a
2.3 7. I
10.0 15.8
7.8 15.4
1.8 11.2
2.3 10.5
4.8 12.9
22:5(n-3) 22:6(n-3) Total (n-3)PUFA"
0.3 1.8 .2.1
2. I 4.7 6.8
0.7 2.5 5.1
2.3 3.7 9.4
2. I 3.4 6.5
0.5 2.8 4.5
18:21n-9) 20:3(n-9) Total PUFA (n-3)/(n-6)
1.3
-
2.2
3.1
2.8
3.9
10.5 0.30
22.6 0.43
22.7 0.33
23.7 0.84
19.8 0.62
21.3 0.35
Notes as described in Table 3.
20:4(n-6) and 22:6(n-3) were the m a j o r fatty acid components of PE, with the percentage of monoenes tending to exceed saturates (Table 4). However saturates, with increased 18:0 and 16:0 were predominant in P S / P I whereas monoenes were lowest in these classes ranging from 25:3 to 39% (Table 5). In all the phospholipid classes, the percentage o f (n-6) P U F A exceeded (n-3)PUFA but the (n-3)/(n-6) ratio was higher than in total lipid. In contrast total neutral lipid generally had a lower total PUFA content and a lower (n-3)/(n-6) ratio than in.total lipid (Table 6). Saturates ranged from 35.8% to 50.2% and predominated over monoenes in the neutral lipid from most cell lines except AS.
Discussion The principal saturated and monounsaturated fatty acids in the cells, 16:0, 18.0, 16:1 and 18: l(n-9) were the same as those normally found in fish tissues
(Ackman 1980; Henderson and Tocher 1987). Similarly 20:4 followed by 18:2 were the principal (n-6)PUFA in the cell lipids as they are in fish tissues (Ackman 1980; Henderson and Tocher 1987). However, although 22:6 was the m a j o r (n-3)PUFA with 22:5 also present, the almost total absence of 20:5 was unexpected, and was a m a j o r qualitative difference between the cultured cells and intact fish tissues. Quantitatively, though, all the cell lines showed considerably lower P U F A levels compared to the tissues of intact fish (Ackman 1980; Henderson and Tocher 1987), ranging from only 9 to 24.9% in the total lipid. These percentages of P U F A were lower than those present in the fetal calf serum, suggesting that the c o m p o n e n t P U F A in the serum may not be ideal for the cells. Significant amounts o f 20:3(n-9) was also detected in the total lipid of most of the cell lines indicating a slight EFA-deficiency. However, 18:2(n-9) was the only (n-9)PUFA detected in TF cells. O f particular interest with respect to fish cells was the very low
225 Table 6. Fatty acid c o m p o s i t i o n o f total n e u t r a l lipids from cell lines Cell line F a t t y acid
RTG-2
TF
AS
CHSE-214
BF-2
FHM
14:0
3.1
1.6
3.6
4.8
5.7
5.7
16:0 18:0
29.2 10.7
27.9 14.9
19.4 7.9
23.1 10.5
24.3 8.7
25.0 I 1.1
Total saturates a
48.5
50.0
35.8
45.3
50.2
49.1
16:1 h
8.6
8.7
10.3
9.8
11.6
8.3
18:1(n-91
19.9
21.1
39.0
16.6
13.8
18.4
18:1(n-71 Total monoencs c
2.9 40.3
2.8 34.8
4.5 56.4
4.5 35.6
2.2 3 I. 1
3.4 33.8
18:21n-61
2.3
2.8
1.4
2.0
2.2
2.0
18:31n-6) 20:3(n-6)
3.9 -
3.8 0.7
0.6 0.6
4.3 2.1
1.9 0.7
2.0 0.7
20:4(n-61 Total (n-6)PUFA d
0.5 6.7
4.1 11.9
1.5 4.1
3.1 I 1.5
4.2 9.0
2.6 7.3
18:3(n-3)
0.3
-
-
0.3
0.2
0.4
22:5(n-3)
(1.2
0.9
0.2
-
O.2
0.3
22:6(n-31 Total (n-3)PUFA e
1.2 1.7
1.5 2.4
1.2 1.4
1.7 2.0
2.0 2.4
1.2 1.9
-
-
-
0.5
2.4
1.5
2.3
6.0
15.9
12.9
11.5
18:2(n-9)
0.8
20:3(n-91 Total PUFA
2.5
-
1(I.9
15.1
(n-3)/(n-61
0.25
0.20
0.34
0.17
0.27
0.26
Notes as d e s c r i b e d in T a b l e 3.
(n-3)PUFA content in total lipid ranging from only 2.1 to 7.8%, which is 5- to 10-fold lower than for normal fish tissues (Ackman 1980; Henderson and Tocher 1987). The distribution of P U F A between the lipid classes was generally similar to that of intact fish tissues with neutral lipids having a lower PUFA content than total lipid and phospholipids (Henderson and Tocher 1987). The (n-3)PUFA were associated more with the phospholipid classes as they all had higher (n-3)/(n-6) ratios than total lipid or total neutral lipid. The highest content of (n-3) PUFA, particularly 22:6(n-3), was found in PE although PC also showed higher (n-3)/(n-6) ratios than total lipid. However, PC was low in PUFA in general and lower than expected. PE and PS can often be more unsaturated than PC in fish tissues (Henderson and Tocher 1987) and this was the situation with the cell lines. The classical 'mammalian' composition of PI with high 18:0 and 20:4(n-6) has been reported for PI in several fish tissues (Bell
et al. 1983; Tocher and Sargent 1984). Although PI
was not fully resolved from PS and therefore not analysed separately in the present study, the P S / P I fractions showed higher 18:0 than PE or PC but 20:4(n-6), although higher than in PC, was generally lower than in PE. Even so, the (n-3)/(n-6)PUFA ratio was generally lower in P S / P I than in either PC. or PE. Table 2 shows that the PUFA level~ and composition of the fish cell lines are partly reflective of the PUFA contained in the fetal calf serum. However, it is notable that the high saturated fatty acid level of the serum is not directly reflected in the total lipid of the cultured cells in which monoenes predominate. This suggests that desaturation and elongation of 14:0 and 16:0 probably occurs resulting in increased percentages of 18:1. Similarly 18:2(n-6) was generally lower in the lipids of the cells than in the serum indicating metabolism either via desaturation/elongation to 20:4(n-6), or via 13oxidation. The significant amounts of 18:3(n-6)
226 and 20:3(n-6) found in all the phospholipid classes in all the cell lines were consistent with the former metabolic pathway. Little 18:3(n-3) was present in the serum or the total lipid from the cultured cells. The (n-3)PUFA are provided by the fetal calf serum primarily as 22:6 and 22:5, and the lack of shorter chain (n-3)PUFA not only reflect this but also indicate little retroconversion occurs. Fetal calf serum did contain approximately 0.6% 20:5(n-3) but this was not selectively incorporated and retained by any cell line as it was only present in a few samples at less 'than 0.5~ There were some variations in the general trends discussed above from one cell type to another. For instance, RTG-2 and TF (the only entirely marine species) were able to maintain the highest total PUFA levels and only these lines, in particular TF, were able to generate an (n-3)/(n-6)PUFA ratio greater than that o[ the serum. Obviously this may partly be a reflection of the lipid class composition of these cells as they also had the highest phospholipid. However, AS cells also contained >60~ phospholipid but were much lower in total PUFA and had a lower (n-3)/(n-6) ratio. In contrast, the AS cells had the highest monoene content of all the cell lines in all the lipid classes. Overall, the results show that fish cell lines grown in culture media supplemented with mammalian sera are deficient in (n-3)PUFA in comparison with tissues from the intact fish. They may also be generally EFA-deficient to some extent. However this has apparently had no major effect on the growth and division of the cells in culture or their long-term survival. For example the RTG-2, BF-2 and FHM cell lines werc all originally established over 20 ),ears ago (Wolf and Quimby 1962; Wolf et al. 1966; Gravell and Malsberger 1965). However culture of fish cells in serum-free or lipid-free media as has been demonstrated with mammalian cells has not been reported for fish cells (Spector et al. 1981). Therefore it remains to be established if fish cells can also grow in the complete absence of all PUFA, both (n-3) and (n-6). It should be noted that incubation temperature is a largely unstudied area in cell culture with respect to lipid metabolism of both mammalian and fish cells (Rosenthal 1987). However a recent study
showed that chick fibroblasts grown in the absence of serum but with stearic acid as a supplement died at 37~ but survived at the slightly higher temperature of 41 ~ (Chester et al. 1986). The inclusion of unsaturated fatty acids prevented the cell death at 37~ This study suggests that incubation temperature plays a major role in survival under conditions where lipid is restricted and therefore should be further studied. This applies particularly to the poikilothermic fish cells which are generally grown at temperatures above the normal temperature range found in the wild, unlike mammalian cells which are generally grown at the physiological temperature of 37~ The fish cells in the present study were grown at 22~ whereas temperatures of 5 - 1 2 ~ are more usual of their various habitats (see Nelson 1984). The cell lines all survive at 12- 15~ where growth, although slower, is not totally arrested (Wolf and Quimby 1967). Cells from some but not all fishes have been grown at temperatures of 5 - 10~ and 'quality of the medium' was reported to be a factor involved (Wolf and Quimby 1969). Physiology of the fish also plays a role; RTG-2 cells grow slowly at temperatures as low as 4~ ( 6 - 7 fold slower than at 20~ whereas FHM cells show little activity at this temperature (Wolf and Quimby 1962; Gravell and Malsberger 1965). Fetal bovine serum was utilized in these studies. Clearly, the slow growth rates and long doubling times of low temperature incubation have resulted in little detailed investigation of the role o f PUFA in cell growth and survival at 5 - 1 0 ~ However, this is an area that requires further investigation to determine the qualitative and quantitative requirements for PUFA, if any, of fish ceils grown at low temperature.
References cited Ackman, R.G. 1980. Fish lipids, part I. In Advances in Fish Science and Technology. pp. 86-103. Edited by J.J. Connell. Fishing News Books Ltd., Farnham, Surrey, U.K. Bailey, J.M. and Dunbar, L.M. 1973. Essential fatty acid requirements of cells in tissue culture: A review. Exp. Mol. Path. 18: 142-161. Bailey, J.M., Howard, B.V., Dunbar, L.M. and Tillman, S.F. 1972. Control of lipid metabolism in cultured cells. Lipids 7: 125-134.
227 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., Pirie, B.J.S. and Sargent, J.R. 1985. Effects of dietary polyunsaturated fatty acid deficiencies on mortality, growth and gill structure in the turbot, Scophthalmus maximus. J. Fish Biol. 26: 181- 191. Burr, G.O. and Burr, M.M. 1930. The nature and role of the fatty acids essential in nutrition. J. Biol. Chem. 86: 587-621. Chester, D.W., Tourtellotte, M.E., Melchior, D.L. and Romano, A.M. 1986. The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function. Biochim. Biophys. Acta 860: 383-398. Christie, W.W. 1982. Lipid Analysis 2nd Edition. Pergamon Press, Oxford. Folch, J., Lees, M. and Sloanc Stanley, G.H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509. Fraser, A.J., Tother, D.R. and Sargent, J.R. 1985. Thin-layer chromatography-flame ionisation detection and the quantitation of marine neutral lipids and phospholipids. J. Exp. Mar. Biol. Ecol. 88: 91-99. Gravell, M. and Malsberger, R.G. 1965. A permanent cell line from the fathead minnow I Pimephalespromelas). Ann. N.Y. Acad. Sci. 126: 555-565. Greene, D.H.S. and Selivonchick, D.P. 1987. Lipid metabolism in fish. Prog. Lipid Res. 26: 53-85. Henderson, R.J. and Tocher, D.R. 1987. The lipid composition and biochemistry of freshwater fish. Prog. Lipid Res. 26: 281-347. Nicholson, B.L. and Byrne. C. 1973. An established cell line from the Atlantic salmon ~Salmo salarL J. Fish. Res. Bd. Can. 30: 913-916. Nelson, J.S. 1984. Fishes o1 The World, 2rid Edition. John Wiley and Sons, New York.
Rosenthal, M.D. 1987. Fatty acid metabolism of isolated mammalian cells. Prog. Lipid Res. 26: 87-124. Sargent, J.R. 1976. The structure, metabolism and function of lipids in marine organisms. In Biochemical and Biophysical Perspectives in Marine Biology. Vol. 3. pp. 149-212. Edited by D.C. Malins and J.R. Sargent. Academic Press, New York. Sargent, J.R. and Whittle, K.J. 1981. Lipids and hydrocarbons in the marine food web. In Analysis of Marine Ecosystems. pp. 491-533. Edited by A.R. Longhurst. Academic Press, London. Sargent, J.R., Henderson, R.J. and Tocher, D.R. 1987. Lipids. In Fish Nutrition, 2nd Edition. Edited by J.E. Halver. Academic Press, New York. (In press). Spector, A.A., Mathur, S.N., Kaduce, T.L. and Hyman, B.T. 1981. Lipid nutrition and metabolism of cultured mammalian cells. Prog. Lipid Res. 19: 155-186. Tinoco, J. 1982. Dietary requirements and functions of r linolenic acid in animals. Prog. Lipid Res. 21: 1-46. 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. Tocher, D.R., Webster, A. and Sargent, J.R. 1986. Utilization of porcine pancreatic phospholipase A 2 for the preparation of a marine fish oil enriched in (n-3) polyunsaturated fatty acids. Biotech. Appl. Biochem. 8: 83-95. Watanabe, T. 1982. Lipid nutrition in fish. Comp. Biochem. Physiol. 73B: 3-15. Wolf, K. and Quimby, M.C. 1962. Established eurythermic line of fish cells in vitro. Science 135: 1065-1066. Wolf, K. and Quimby, M.C. 1967. Low-temperature incubation using a water supply. Appl. Microbiol. 15: 1501. Wolf, K. and Quimby, M.C. 1969. Fish cell and tissue culture. In Fish Physiology. Vol. III. pp. 253-305. Edited by W.S. Hoar and D.J. Randall. Academic Press, New York. Wolf, K., Gravell, M. and Malsberger, R.G. 1966. Lymphocystis virus: Isolation and propagation in centrarchid fish cell lines. Science 151: 1004-1005.
The fatty acid compositions of established fish cell lines after long-term culture in mammalian sera D.R. Tocher ~, J.R. Sargent I and G.N. Frerichs 2 I N E R C Unit o f Aquatic Biochemistry, Dept o f Biological Science, Universitv q f Stirling, Stirling FK9 4LA, Scotland, U.K. 2 Institute o f Aquaculture, University o f Stirling, Stirling FK9 4LA, Scotland, U.K. Keywords: fatty acids, compositions, fish cells, culture
Abstract The effect of long-term culture of fish cells in mammalian serum on the phospholipid fatty acid composition was investigated. All the cell lines studied had much lower levels of polyunsaturated fatty acids (PUFA) than those found in intact fish tissues. In particular (n-3)PUFA were considerably depleted in the cultured cell lines, leading to very low (n-3)/(n-6) ratios in all the phospholipid classes. In general the cells were rich in 18:1, 16:0, 18:0 and 16:1 with 20:4(n-6) and 22:6(n-3) as the major PUFA. The fatty acid composition reflected the composition o f the fetal calf serum added to the media rather than their fish tissue origins. The results were discussed in relation to the roles of PUFA in general cell metabolism and more specifically the role of (n-3)PUFA in fish cells.
Abbreviations PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PUFA, polyunsaturated fatty acids.
Introduction Whereas the PUFA of terrestrial mammals, including man, are dominated by the (n-6) series, in aquatic and especially marine organisms, (n-3) PUFA predominate (Sargent 1976; Sargent and Whittle 1981; Sargent et al. 1987). This is perhaps reflected in the qualitatively different essential fatty acid requirements for mammals and fish. Mammals have an absolute requirement for 18:2(n-6) (Burr and Burr 1930), whereas their requirement for 18:3(n-3) has been difficult to establish (Tinoco 1982). In contrast, fish have a well-defined require-
merit for 18:3(n-3) (Watanabe 1982; Greene and Selivonchick 1987) and also long chain (n-3)PUFA in the case of some marine fish (Watanabe 1982; Greene and Selivonchick 1987; Sargent et al. 1987) whereas the qualitative and quantitative requirements for 18:2(n-6) or other (n-6)PUFA have not been conclusively established (Watanabe 1982; Bell' et al. 1985; Sargent et al. 1987; Henderson and Tocher, 1987). It has been reported, however, that the majority of mammalian cell lines that have been investigated appear to grow well in the absence of PUFA, both essential and non-essential (Bailey and Dunbar 1973). However the authors of that study also observed that significant amounts of linoleic acid were consistently found in samples of commercially obtained chemically defined 'lipid-free' culture media (Bailey et al. 1972). It is routine for most tissue culture media to contain no lipid or essential fatty acids other than those present in the serum supple-
220 ment. The lipids and fatty acids thus available to tissue culture cells are dependent upon the type of serum utilized in the medium. Fish cell culture was fi'rst established almost 30 years ago and, as with mammalian cells, serum was found to be an essential supplement (Wolf and Quimby 1969). Sera from many species, including homologous and heterologous fish, have proved very variable in growth promoting properties (Wolf and Quimby 1969). Furthermore, the considerable difficull, ies in obtaining sufficient fish serum for cell culture combined with the risk of introducing latent fish viruses has determined that fetal calf serum has been the serum of choice for fish cell culture as with mammalian cell culture. In this study we have determined the fatty acid profile of a variety of teleost fish cell lines to investigate the effect that long-term culture in mammalian serum has had on PUFA levels, and particularly the (n-3)PUFA content. In this report we compare and discuss the fatty acid compositions of the different phospholipid classes in the various fish cell lines.
Materials and methods
Cell lines Rainbow trout (Sahno gairdneri) gonad (RTG-2) (Wolf and Quimby 1962), bluegill (Lepomis macrochirus) fry (BF-2) (Wolf et al. 1966), fathead minnow (Pimephales promelas) (FHM) (Gravell and Malsberger 1965) and Atlantic salmon (Salmo salar) (AS) (Nicholson and Byrne 1973) cell lines were obtained originally from Flow Laboratories, Rickmansworth, U.K. The chinook salmon (Oncorhynchus tshawytscha) epithelium cell line (CHSE-214) was obtained from the Microbiology section, Institute of Aquaculture, University of Stifling. All these cell lines had been maintained in continuous culture in the Institute of Aquaculture for many passages. The turbot (Scophthalmus maximus) fin cell line (TF) was a gift from Dr. B. Hill, MAFF Fish Diseases Laboratory, Weymouth, U.K., who had originally established the line.
Media and growth conditions AS, BF-2 and CHSE-214 cells were grown in minimum essential medium Eagle (modified) with Earle's salts containing 0.25~ bicarbonate and 1% non-essential amino acids. RTG-2 cells were grown in Glasgow's modification of Eagle's medium containing 16 mM Tris-HC1 buffer (Trizma, pH 7.4), 0.3~ sodium bicarbonate and 10~ tryptose phosphate broth. Both FHM and TF cells were grown in Leibovitz L-15 medium, with 0.35~ sodium chloride added in the case of TF cells. All media were further supplemented with 10070 fetal calf serum and antibiotics (50 I.U./ml penicillin and 50 ~g/ml streptomycin). The cell cultures were grown to confluence in sealed 75 cm: plastic flasks (Gibco Ltd., Paisley, U.K.) at 22~
Cell harvesting and lipid extraction Cells were harvested within 24 h of reaching confluence. Media were decanted from the flasks and the monolayers washed with phosphate buffered saline before addition of 1 ml Trypsin-EDTA (0.05~ and 0.02~ respectively). When the cells were dissociated from the substrate, 2 ml of the appropriate medium were added to inhibit further action of the trypsin. The cells were centrifuged at 600 • g for 10 mins at 4~ the supernatant poured off and the cell pellet washed 3 times with 10 ml phosphate buffered saline to ensure removal of all serum. Total lipid was extracted from the washed cells essentially according to Folch et al. (1957) by addition of 5 ml ice cold c h l o r o f o r m / m e t h a n o l (2:1, v/v) containing 0.05~ butylated hydroxytoluene. After homogenisation in a glass-teflon pestle homogeniser, the extract was filtered and 1 ml 0.88o7o KCI added. Following mixing and separation of the phases by centrifugation, the chloroform layer was taken and dried down under nitrogen before being redissolved in c h l o r o f o r m / methanol (2:1, v/v) containing 0.05~ butylated hydroxytoluene at a final lipid concentration of I00 mg/ml.
221
Lipid and .fatty acid analysis Lipid class analysis was performed by thin-layer chromatography-flame ionisation detection (TLCFID) using an latroscan TH-10 mk.lV (latron Laboratories, Tokyo) equipped with a HewlettPackard HP3300 recording integrator. Precise details of methodology and calibration of the instrument have been described previously (Fraser et al. 1985). Total lipid was separated into individual neutral and phospholipid classes by l-dimensional and 2-dimensional TLC, respectively, as described previously (Tocher and Sargent 1984). Fatty acid methyl esters were prepared by acid-catalysed transmethylation (Christie 1982) with subsequent purification of the extracted methyl esters by TLC. The methyl esters were analysed on a Packard 436 gas chromatograph (Packard Instruments Inc., Caversham, U.K.) equipped with a chemically bonded CP Wax 52CB fused silica capillary column (50 m • 0.34 mm i.d.) (Chrompack U.K. Ltd., London), an on-column injector and using H, as carrier gas. Details of operating conditions and identification of fatty acid methyl esters were as described previously (Tocher and Sargent 1984; Tocher et al. 1986). Data are means of triplicate GC analyses; standard deviations are omitted for clarity but were generally less than 507o.
Materials All media, phosphate buffered saline, sodium bicarbonate, non-essential amino acids, tryptose phosphate broth, antibiotics, fetal call" serum and trypsin-EDTA were obtained from Flow Laboratories, Rickmansworth, U.K. Butylated hydroxytoluene and Trizma were from Sigma Chemical Co. Ltd., Poole, U.K. All solvents were H P L C grade and were obtained from Rathburn Chemicals, Walkerburn, U.K.
Results
All the cell lines used in the present study were known to have been maintained in culture media
containing mammalian sera, almost exclusively fetal calf serum, for many passages prior to the onset of the experiment, and for 3 passages during the experiment before harvesting. Phospholipids accounted for from over 72% to under 41% of the total lipid, decreasing in the order RTG-2 > TF > AS > FHM > BF-2 > CHSE-214 (Table 1). Triacylglycerol content was inversely related to the total phospholipid and increased in the cells in the same order as above, rising from 2.2% in RTG-2 cells to 28.4% in CHSE-214. Sterol esters varied and did not relate to the neutral lipid or triacylglycerol levels. Cholesterol was more constant averaging approximately 19% in most cell lines other than FHM which had 12.4%. Phosphatidylcholine (PC) was the major phospholipid class in all cases but was highly variable ranging from 19.4% to 40.8% of total lipid, whereas phosphatidylethanolamine (PE) was more constant at around 20% (Table 1). RTG-2 cells were notable for their high content (11.1~ of phosphatidylserine (PS)/phosphatidylinositol (PI) in comparison with the other cells. Fetal calf serum lipid was primarily sterol ester and triacylglycerol with lesser amounts of PC, sphingomyelin and free cholesterol. In total lipid, monoenes (primarily 18:1(n-9)) accounted for approximately 40%-60~ of the total fatty acids with saturates (primarily 16:0 and 18:0) accounting for between 27% and 36% (Table 2). Total PUFA was highest in RTG-2 and TF at almost 25~ and 24% respectively but was only 9% in AS and BF-2. The (n-6)PUFA predominated over (n-3)PUFA by 2-fold (RTG-2) to over 3-fold ' (CHSE-214). Arachidonic acid (20:4).and 22:6 were the major (n-6) and (n-3)PUFA respectively. Of the individual phospholipid classes, PC was lowest in PUFA, having levels lower than those in total lipid ranging from 8.2% to 12.0070 (Table 3) with CI8 PUFA more prominent as compared with total lipid. Monoenes were slightly lower in PC than in total lipid but saturates were considerably higher, due to increased 16:0. The highest levels of PUFA were found in PE and P S / P i in all cell lines (Tables 4 and 5). In PE and P S / P I , total PUFA ranged from 21.9 to 36.2% and from 10.5 to 23.7%, respectively. In general 18: l(n-9), 16:0, 18:0, 16:1,
222
Table I. Lipid class composition of the cell lines and fetal calf serum (FCS) Cell
line
Lipid
RTG-2
TF
AS
CHSE-214
% Phospholipids
72.4
65.8
61.7
40.5
46.8
56,1
13,2
% Neutral lipids
27.6
34.2
38,3
59.5
53.2
43.9
86.8
36.7 21.5
35.4 23.7
40.8 19.4
19.4 17.3
24.7 19.7
31. l 21,1
8.2 -
I I. I 3. I
3.5 3. I
0,4 I. I
1,0 2.8
0.8 1,6
1.4 2.5
4.8
2.2 2.2 1.8 21.3
9.9 4.6 2.6 17.2
3.2 13.9 2.3 18.9
9.7 28.4 2.0 19.3
5,6 24.7 3.7 19.0
8.4 21.3 1.7 12.4
58. I 24.2 0.8 3.7
Phospholipid classes: (% total lipid) Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine/ inositol Sphingomyelin Neutral classes: total lipid) Sterol ester Triacylglycerol Free fatty acid Cholesterol
BF-2
FHM
FCS
(%
-
= not detected.
Table 2. Fatty acid compositions of total lipid from cell lines and fetal calf serum Cell line Fatty acid
RTG-2
TF
AS
CHSE-214
BF-2
FHM
FCS r
14:0 16:0 18:0 Total saturates a
1.2 13.8 9.6 27.0
2.2 17.7 12.8 36.4
1.7 16.1 6.6 26.7
1.4 15.0 7.3 26.6
1.1 18.6 11.7 35.6
1.2 15.8 9.7 29.4
2.6 22.1 11.1 39.3
16:1 b, 18:1(n-9) 18:1(n-7) Total monoenes c
8.7 32.6 4.5 47.9
10.1 24.3 3.9 38.9
9.6 45.7 3.8 61.0
10.5 25.5 5.4 50.4
15.3 21.9 4.0 43.9
9.8 33.7 5.7 52.2
5.2 18.7 5.6 31.9
18:2(n-6) 18:3(n-6) 20:2(n-6) 20:3(n-6) 20:4(n-6) Total (n-6)PUFA d
1.8 1.4 1.8 1.6 8.4 16. I
2.5 0.5 0.2 1.5 8.6 15.9
1.5 0.3 0.2 0.6 3.4 6.3
1.8 0.4 0.7 1.4 5.2 9.9
0.8 0.6 0.6 0.2 4.0 6.4
1.2 0.1 2.0 1.5 4.8 9.9
4.7 0.2 0.3 1.9 8.9 16.6
-
-
-
0.3
0.5
0.5
0.3
0.2
-
-
2.3 3.4 6.4
2.3 5.2 7.8
0.9 1.1 2.2
1.0 2.0 3.0
18:2(n-9)
-
1.0
.
20:3(n-9)
2.4
-
0.5
1.8
24.9 0.40
24.7 0.49
9.0 0.35
14.7 0.30
18:3(n:.3) 20:5{n-3) 22:5(n-3) 22:6(n-3) Total (n-3)PUFA c
Total PUFA (n-3)/(n-6)
0.2
.
-
0.3 1.8 2. I .
0.6
1.1 2.0 3.4
2.2 3.3 6.6
0.5
3.4
0.2
9.0 0.33
16.7 0.34
24.0 0.40
.
.
Results are expressed as a % of weight: aalso includes 15:0, 17:0, 20:0 and 22:0; bpredominantly (n-7) isomer: ':also includes 20:1 isomers, 22: I and 24. I : dalso includes 22:4(n-6); Calso includes 18:4(n-3) and 20:4(n-3); raverage of the two batches of FCS used in this study; - = not detected.
223
Table 3. Fatty acid composition of phosphatidylcholine from cell lines Cell line Fatty acid
RTG-2
TF
AS
CHSE-214
BF-2
FHM
14:0 16:0 18:0 Total saturates ~'
5.2 28.2 9.8 52.6
4.5 26.2 7.3 43.9
3.0 21.8 5.8 35.3
1.8 23.2 7.6 39.1
3.0 25.7 7.3 43.3
2.7 23.7 8.0 40.7
16:1 b 18: I (n-9) 18: I (n-7) Total monoenes c
13.4 19.5 2.1 36.0
14.3 23.8 3.2 42.6
13.0 38.9 3.5 56.1
10.8 31. I 4.4 48.6
20.0 24.0 3.0 48.3
13.7 32.0 3.8 50.5
18:2(n-6) 18:3(n-6) 20:3(n-6) 20:4(n-6) Total (n-6)PUFA a
1.8 2.8 1.4 6.0
2.3 1.3 0.9 3.2 8.1
2. I 1.2 0.2 0.7 4.2
3.0 1.9 1.4 1.4 7.7
2.5 0.9 0.3 0.8 4.5
2.1 1.0 0.6 1.0 4.7
22:501-3) 22:6(n-3) Total (n-3)PUFA ~'
O. I 3.4 3.5
0.5 2.9 3.4
0.2 I. I 4.0
0.1 1.6 3.3
0. I 2.0 3.4
1.8 2.4
0.2
. 1.0
0.3
1.6
8.4 0.95
12.0 0.43
8.2 0.78
8.7 0.51
18:2(n-9) 20:3(n-9)
.
Total PUFA (n-3)/(n-6)
9.5 0.58
I 1.5 0.42
.
.
Notes as described in Table 2 except ':also includes 18:3(n-3), 18:4(n-3), 20:4(n-3) and 20:5(n-3).
Table 4. Fatty acid composition of phosphatidylethanolamine from cell lines Cell line Fatty acid
RTG-2
TF
AS
CHSE-214
BF-2
FHM
14:0 16:0 18:0 Total saturates a
4.9 13.8 10.4 36.7
1.9 12.4 13.4 32.2
1.9 13.2 5.6 26.5
1.0 12.0 7.6 26.0
2.9 16.0 I 1.8 36.5
1.7 1I.I 8.8 26.9
16:1 t' 18: l(n-9) 18:1(n-7) Total monoenes c
9.7 19.6 1.5 33.3
7.7 16. I 3.9 28.5
12.3 34.7 2.6 50.8
7.6 23.8 5.9 45.4
13.9 17. I 4.2 37.9
8.4 27.0 " 5.2 43.8
18:2(n-6) 18:3(n-6) 20:2(n-6) 20:3(n-6) 20:4(n-6) Total (n-6)PUFA d
3.0 1.3 I.I 0.6 10.3 16.5
2.7 1.8 0.5 1.4 14.4 23.6
3.8 1.9 0.4 0.9 5.1 12.4
3.5 2.1 0.5 1.8 6.7 15.6
2.7 3.5 0.8 0.8 3.9 12.9
2.7 I.I 3.6 1.8 7.4 17.2
22:5(n-3) 22:6(n-3) Total (n-3)PU FA c
2.0 5.5 7.5
3.0 7.4 12.0
1.5 3.9 7.6
1.8 4.6 9.6
1.8 4.7 9.2
1.4 3.8 7.0
18:2(n-9) 20:3(n-9)
1.9
0.6 -
. 1.9
3. I
1.5
4.8
Total PUFA (n-3)/(n-6)
25.9 0.46
36.2 0.51
21.9 0.61
28.3 0.62
23.6 0.71
29.0 0.41
Notes as described in Table 3.
.
.
.
224 Table 5. Fatty acid composition of phosph/uidylserirle/phosphatidylinositol from cell lines Cell line Fatty acid
RTG-2
TF
AS
CHSE-214
BF-2
FHM
14:0 16:0 18:0 Total saturales a
5.3 25.2 13.2 51.8
3.6 20.7 19.9 50.6
1.5 16.5 13.3 38.0
6.8 19.1 I 1.9 45.8
2.4 19.6 17.3 49.0
3.8 20.7 14.1 45.0
16:1 b 18:1(n-9) 18:11n-7) Total mofioencs c
I 1.4 13.5 1.8 28.3
8.9 13.8 1.4 25.6
9.2 24.5 2.2 39.0
9.6 12.1 1.9 25.3
11.8 14.9 I.I 29.0
2.3 16+1 1.7 32.3
18:2(n-6)
2.1
1.4
2.5
2.9
3.1
2.6
18:31n-61 20:2(n-61 20:31n-6)
1.8 0.6
1.3 0.2 1.2
2.1 .0.8 1.8
3.9 0.8 1.8
3.4 0.6 1.0
1.3 2.8 1.3
20:41n-61 Total (n-6)PU FA a
2.3 7. I
10.0 15.8
7.8 15.4
1.8 11.2
2.3 10.5
4.8 12.9
22:5(n-3) 22:6(n-3) Total (n-3)PUFA"
0.3 1.8 .2.1
2. I 4.7 6.8
0.7 2.5 5.1
2.3 3.7 9.4
2. I 3.4 6.5
0.5 2.8 4.5
18:21n-9) 20:3(n-9) Total PUFA (n-3)/(n-6)
1.3
-
2.2
3.1
2.8
3.9
10.5 0.30
22.6 0.43
22.7 0.33
23.7 0.84
19.8 0.62
21.3 0.35
Notes as described in Table 3.
20:4(n-6) and 22:6(n-3) were the m a j o r fatty acid components of PE, with the percentage of monoenes tending to exceed saturates (Table 4). However saturates, with increased 18:0 and 16:0 were predominant in P S / P I whereas monoenes were lowest in these classes ranging from 25:3 to 39% (Table 5). In all the phospholipid classes, the percentage o f (n-6) P U F A exceeded (n-3)PUFA but the (n-3)/(n-6) ratio was higher than in total lipid. In contrast total neutral lipid generally had a lower total PUFA content and a lower (n-3)/(n-6) ratio than in.total lipid (Table 6). Saturates ranged from 35.8% to 50.2% and predominated over monoenes in the neutral lipid from most cell lines except AS.
Discussion The principal saturated and monounsaturated fatty acids in the cells, 16:0, 18.0, 16:1 and 18: l(n-9) were the same as those normally found in fish tissues
(Ackman 1980; Henderson and Tocher 1987). Similarly 20:4 followed by 18:2 were the principal (n-6)PUFA in the cell lipids as they are in fish tissues (Ackman 1980; Henderson and Tocher 1987). However, although 22:6 was the m a j o r (n-3)PUFA with 22:5 also present, the almost total absence of 20:5 was unexpected, and was a m a j o r qualitative difference between the cultured cells and intact fish tissues. Quantitatively, though, all the cell lines showed considerably lower P U F A levels compared to the tissues of intact fish (Ackman 1980; Henderson and Tocher 1987), ranging from only 9 to 24.9% in the total lipid. These percentages of P U F A were lower than those present in the fetal calf serum, suggesting that the c o m p o n e n t P U F A in the serum may not be ideal for the cells. Significant amounts o f 20:3(n-9) was also detected in the total lipid of most of the cell lines indicating a slight EFA-deficiency. However, 18:2(n-9) was the only (n-9)PUFA detected in TF cells. O f particular interest with respect to fish cells was the very low
225 Table 6. Fatty acid c o m p o s i t i o n o f total n e u t r a l lipids from cell lines Cell line F a t t y acid
RTG-2
TF
AS
CHSE-214
BF-2
FHM
14:0
3.1
1.6
3.6
4.8
5.7
5.7
16:0 18:0
29.2 10.7
27.9 14.9
19.4 7.9
23.1 10.5
24.3 8.7
25.0 I 1.1
Total saturates a
48.5
50.0
35.8
45.3
50.2
49.1
16:1 h
8.6
8.7
10.3
9.8
11.6
8.3
18:1(n-91
19.9
21.1
39.0
16.6
13.8
18.4
18:1(n-71 Total monoencs c
2.9 40.3
2.8 34.8
4.5 56.4
4.5 35.6
2.2 3 I. 1
3.4 33.8
18:21n-61
2.3
2.8
1.4
2.0
2.2
2.0
18:31n-6) 20:3(n-6)
3.9 -
3.8 0.7
0.6 0.6
4.3 2.1
1.9 0.7
2.0 0.7
20:4(n-61 Total (n-6)PUFA d
0.5 6.7
4.1 11.9
1.5 4.1
3.1 I 1.5
4.2 9.0
2.6 7.3
18:3(n-3)
0.3
-
-
0.3
0.2
0.4
22:5(n-3)
(1.2
0.9
0.2
-
O.2
0.3
22:6(n-31 Total (n-3)PUFA e
1.2 1.7
1.5 2.4
1.2 1.4
1.7 2.0
2.0 2.4
1.2 1.9
-
-
-
0.5
2.4
1.5
2.3
6.0
15.9
12.9
11.5
18:2(n-9)
0.8
20:3(n-91 Total PUFA
2.5
-
1(I.9
15.1
(n-3)/(n-61
0.25
0.20
0.34
0.17
0.27
0.26
Notes as d e s c r i b e d in T a b l e 3.
(n-3)PUFA content in total lipid ranging from only 2.1 to 7.8%, which is 5- to 10-fold lower than for normal fish tissues (Ackman 1980; Henderson and Tocher 1987). The distribution of P U F A between the lipid classes was generally similar to that of intact fish tissues with neutral lipids having a lower PUFA content than total lipid and phospholipids (Henderson and Tocher 1987). The (n-3)PUFA were associated more with the phospholipid classes as they all had higher (n-3)/(n-6) ratios than total lipid or total neutral lipid. The highest content of (n-3) PUFA, particularly 22:6(n-3), was found in PE although PC also showed higher (n-3)/(n-6) ratios than total lipid. However, PC was low in PUFA in general and lower than expected. PE and PS can often be more unsaturated than PC in fish tissues (Henderson and Tocher 1987) and this was the situation with the cell lines. The classical 'mammalian' composition of PI with high 18:0 and 20:4(n-6) has been reported for PI in several fish tissues (Bell
et al. 1983; Tocher and Sargent 1984). Although PI
was not fully resolved from PS and therefore not analysed separately in the present study, the P S / P I fractions showed higher 18:0 than PE or PC but 20:4(n-6), although higher than in PC, was generally lower than in PE. Even so, the (n-3)/(n-6)PUFA ratio was generally lower in P S / P I than in either PC. or PE. Table 2 shows that the PUFA level~ and composition of the fish cell lines are partly reflective of the PUFA contained in the fetal calf serum. However, it is notable that the high saturated fatty acid level of the serum is not directly reflected in the total lipid of the cultured cells in which monoenes predominate. This suggests that desaturation and elongation of 14:0 and 16:0 probably occurs resulting in increased percentages of 18:1. Similarly 18:2(n-6) was generally lower in the lipids of the cells than in the serum indicating metabolism either via desaturation/elongation to 20:4(n-6), or via 13oxidation. The significant amounts of 18:3(n-6)
226 and 20:3(n-6) found in all the phospholipid classes in all the cell lines were consistent with the former metabolic pathway. Little 18:3(n-3) was present in the serum or the total lipid from the cultured cells. The (n-3)PUFA are provided by the fetal calf serum primarily as 22:6 and 22:5, and the lack of shorter chain (n-3)PUFA not only reflect this but also indicate little retroconversion occurs. Fetal calf serum did contain approximately 0.6% 20:5(n-3) but this was not selectively incorporated and retained by any cell line as it was only present in a few samples at less 'than 0.5~ There were some variations in the general trends discussed above from one cell type to another. For instance, RTG-2 and TF (the only entirely marine species) were able to maintain the highest total PUFA levels and only these lines, in particular TF, were able to generate an (n-3)/(n-6)PUFA ratio greater than that o[ the serum. Obviously this may partly be a reflection of the lipid class composition of these cells as they also had the highest phospholipid. However, AS cells also contained >60~ phospholipid but were much lower in total PUFA and had a lower (n-3)/(n-6) ratio. In contrast, the AS cells had the highest monoene content of all the cell lines in all the lipid classes. Overall, the results show that fish cell lines grown in culture media supplemented with mammalian sera are deficient in (n-3)PUFA in comparison with tissues from the intact fish. They may also be generally EFA-deficient to some extent. However this has apparently had no major effect on the growth and division of the cells in culture or their long-term survival. For example the RTG-2, BF-2 and FHM cell lines werc all originally established over 20 ),ears ago (Wolf and Quimby 1962; Wolf et al. 1966; Gravell and Malsberger 1965). However culture of fish cells in serum-free or lipid-free media as has been demonstrated with mammalian cells has not been reported for fish cells (Spector et al. 1981). Therefore it remains to be established if fish cells can also grow in the complete absence of all PUFA, both (n-3) and (n-6). It should be noted that incubation temperature is a largely unstudied area in cell culture with respect to lipid metabolism of both mammalian and fish cells (Rosenthal 1987). However a recent study
showed that chick fibroblasts grown in the absence of serum but with stearic acid as a supplement died at 37~ but survived at the slightly higher temperature of 41 ~ (Chester et al. 1986). The inclusion of unsaturated fatty acids prevented the cell death at 37~ This study suggests that incubation temperature plays a major role in survival under conditions where lipid is restricted and therefore should be further studied. This applies particularly to the poikilothermic fish cells which are generally grown at temperatures above the normal temperature range found in the wild, unlike mammalian cells which are generally grown at the physiological temperature of 37~ The fish cells in the present study were grown at 22~ whereas temperatures of 5 - 1 2 ~ are more usual of their various habitats (see Nelson 1984). The cell lines all survive at 12- 15~ where growth, although slower, is not totally arrested (Wolf and Quimby 1967). Cells from some but not all fishes have been grown at temperatures of 5 - 10~ and 'quality of the medium' was reported to be a factor involved (Wolf and Quimby 1969). Physiology of the fish also plays a role; RTG-2 cells grow slowly at temperatures as low as 4~ ( 6 - 7 fold slower than at 20~ whereas FHM cells show little activity at this temperature (Wolf and Quimby 1962; Gravell and Malsberger 1965). Fetal bovine serum was utilized in these studies. Clearly, the slow growth rates and long doubling times of low temperature incubation have resulted in little detailed investigation of the role o f PUFA in cell growth and survival at 5 - 1 0 ~ However, this is an area that requires further investigation to determine the qualitative and quantitative requirements for PUFA, if any, of fish ceils grown at low temperature.
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