Journal ofhieurochemistry. 1975, Vol. 24, pp 71-77. Pergamon Press. Printed in Great Britain.

POLYUNSATURATED FATTY ACID METABOLISM IN NEUROBLASTOMA CELLS IN CULTURE’ E. YAWN,’ZIVAYAVIN’ and J. H. MENKES~ Brentwood V.A. Hospital Los Angeles, the Cedars-Sinai Medical Center and the Division of Pediatric Neurology, University of California a t Los Angeles, CA 90073, U.S.A. (Received 4 June 1974. Accepted 13 June 1974)

Abstract-Neuroblastoma cell cultures took up linoleic and linolenic acids at approximately equal rates, and incorporated them into a variety of lipid fractions, principally cellular phospholipids. Linoleic acid was preferentially incorporated into choline phosphoglycerides, while most of the radioactivity derived from linolenic acid entered ethanolamine phosphoglycerides. There was no evidence for direct transfer of fatty acids between these two phosphoglyceride fractions. When, after the addition of cytosine arabinoside, cell division was arrested, the entry of labelled fatty acids into ethanolamine and serine phosphoglycerides was reduced, suggesting that these lipids are involved in the formation of new cell membranes. In the ethanolamine phosphoglyceride fraction, phosphatidal ethanolamine (plasmalogen) was the principal acceptor for the higher polyunsaturated fatty acids of the 0 3 series. The ratio of labelled fatty acids entering ethanolamine plasmalogens to that entering ethanolamine phosphoglycerides increased following the addition of cytosine arabinoside, suggesting plasmalogens to be involved in formation of cell processes. The first step in the metabolism of both linoleic and linolenic acid was the addition of a two-carbon unit. Conversion of linoleic acid to higher polyunsaturated fatty acids was slower than the conversion of linolenic acid to its higher analogues. This contrasted with the behaviour of dissociated cultures of normal brain cells which were able to form higher analogues of linoleic and linolenic acids at nearly equal rates.

REMOVALof serum from the medium of neuroblastoma any alteration in the metabolism of essential fatty acids cultures or addition of dBc AMP induces transforma- accompanying morphological transformation, and no tion of the cells from a spherical nondifferentiated form indication whether differences between malignant and to a state approximating a differentiated neuron (SEEDS non-malignant tissue are due to active cell division et al., 1970, PRASAD & HSIE,1971, FURMANSKI et al., characteristic for the malignant state, or due to a loss 1971). Withdrawal of serum promotes growth of pro- of discrete metabolic capacities. cesses or neurites up to several hundred microns in The literature on the differences between lipid metalength, and halts cellular proliferation, suggesting that bolism of normal and neoplastic tissue is too extensive the cells have assumed less malignant characteristics. to be covered at this point; the reader is referred to an These morphological changes are accompanied by in- excellent review by BERGELSON (1972). The metabolism duction of enzymatic (BLUME et al., 1970) and electro- ofessential fatty acids in primary heart cultures and in physiological (PEACOCK et al., 1972) activities charac- cell lines of neoplastic origin has been studied by teristic of neurons. There are, however, no reports on several groups (MEAD& HAGGARTY, JR., 1969, HARARY et al., 1967; BAILEY et al., 1972). Both types of cell Supported by Research Grants NB 06938, and CA Systems require essential fatty acids for normal oxida13538 from the National Institutes of Health and by funds tive phosphorylation. Freshly isolated heart cells conof the National Genetics Foundations, Inc. and Children’s vert linoleic acid (18 :2) to arachidonic acid (20:4), a Brain Diseases. metabolic capability lost in the course. of culture, and Present address: Dept. of Biochemistry, Weizmann Inst. not demonstrable in H ~ cells,~EWHoUsE L ~ et al. (1953) Rehovot, Israel. have shown that in comparison with non-neoplastic Present address: Dr. J. H. Menkes, 9615 Brighton Way, tissue, many tumours have a reduced capacity to synBeverly Hills, CA 90210, U S A . fatty acids from precursors such as glucose or Abbreviations used: d&, dibutyryl cyclic AMP; CPG, choline phosphoglyceride; EPG, ethanolamine phosphogly- acetate. Therefore Some of the fatty acids required by ceride; SPG serine phosphoglyceride; DPG, diphosphatidyl neoplastic tissue are Probably transported from other glycerol. tissues in oiuo,or are present in the serum added to the 71

72

E. YAWN,Z. YAVINand J. H. MENKES

culture medium when t u m o u r s a r e g r o w n in u i t r n Stein and coworkers found that brain t u m o u r s have a high percentage of docosahexaenoic acid (22 :6), a n d a l o w percentage of linoleic acid (1 8 :2). when c o m p a r e d with normal brain lipids (STEINr t al., 1965). The ability to induce morphological a n d biochemical changes within the same cell population p r o m p t e d us t o study the neuroblastoma model, with the a i m of obtaining information on the role of essential fatty acids in tumor metabolism under various conditions of cellular differentiation. MATERIALS A N D METHODS Growth and inaitittwuncr of tieurohlastotna crlls. A clonal cell line of mouse netiroblastoma C1300 (Clonc N-18) (Gift of Dr. H. Herschman, UCLA) was used for the routine experiments. Cells were grown in plastic petri dishes (60 mm, Falcon Plastic Corp.) in Dulbecco's modified Eagle's medium (Microhiol. Assoc. Inc., Bethesda, MD) containing 10% fetal calf serum (Rehatuin-Reheis Chem. Corp.. Chicago) and a penicillin-streptomycin solution (0.25 mg each per 100 ml medium). Cells were maintained at 37°C in an atmosphere of 957; air and 5'7" C O L ,and transferred every 3-4 days to maintain logarithmic growth. Cells were tested for mycoplasma and found free of any detectable contamination. Incorporution studies. Cells in the logarithmic phase were routinely transferred by treating them with 0.25% Viokase (Grand Island Biol. Co., Grand Island, NY) and plated in 60 mm petri dishes at a density of 5-10 x lo-' cells per dish, containing 2 ml medium. After 48 h the medium was removed and two groups of cultures were treated with either Nh,O' -dibutyryl cyclic AMP (1 x lo-' M, Sigma) or cytosine arabinoside (2 x lo-' mM, Sigma), both added in 10% serum-containing medium. One group of petri dishes was incubated with serum-free medium and a fourth group, incubated in medium containing lo:{ fetal calf serum, but no additives, served a s control. After 48 h at 37"C, the preincubation medium was replaced by medium of thc same constitution hut containing radioisotopes. [ I-'4C]linoleic acid (57 mCi/mmol, Amersham-Searle) and [ I-'4C]linolenic acid (52.4 mCi/mmol, Amersham-Searle) were complexed with fatty acid-free albumin (Pentex, Miles Lab.), added to the medium, and incubated with the cells as previously described (YAWN& MENKES,1973b). C6-astrocytoma cell line was grown in Ham's F-10 medium with 10% fetal calf serum. Cultured dissociated brain cells were prepared according to YAWN & MENKES ( 1973~). Extraction ofcullular lipids. Procedural details have been described elsewhere (YAWN& MENKES,1973h). In essence, the washed cell pellet of 2 to 4 combined petri dishes was resuspended in cold saline. Eight vol of chloroform:methanol (1:2, v/v) were added and cells were briefly homogenized with a Potter--Elvehjem homogenizer. The lipid extract was allowed to remain for 2 h at 4°C or overnight a t -20°C. It was then centrifuged for 10 min at 1600 g and the organic

phase transferred to another tube to which chloroform and water were added to obtain a final ch1oroform:methanol:water ratio of8:4:3 (by vol.). The ensuing aqueous phase was removed and the lipid classes separated by T L C (YAWN & MENKES, 1973b). The DNA content of the cells was deter& AGKANOFF mined by the procedure described by SANTEN (1963). The amount of DNA obtained from each petri dish ranged from 50 to 80 pg, Separatioti ofthr lipid clusst,s. Analysis of the radioactivity incorporated into the various phospholipids was carried out using a modification of the two dimensional T L C method described by ROUSERet al. (1970). Aliquots of the lipid extract were spotted on 10 x 10 cm precoated silica Gel G (Merck, Darmstadt) T L C plates and developed in cbloroform :methanol : 28% aqueous ammonia (65 :35 : 5, by vol.). In order to separate the diphosphatidyl glycerol, phosphatidal ethanolamine and phosphatidyl cthanolamine glycerides, the lipid lane developed in the first dimension was exposed for 3 min to HCI fumes to cleave the vinyl ether & SUN.1972). Plates were then dried for bonds (HORROCKS 10 min under a stream of air and developed in the second solvent system which consisted of chloroform: acetone: methanol: acetic acid: water ( 1 0 : 4 : 2 : 2 :I , by vol.). Plates were dried and scanned for rddiodctivity using a BertholdVarian Aerograph radioscanner, Model 6000. Alternatively the lipids were exposed to iodine vapours and the spots were & MENKES,1973h). scraped off the plate and counted (YAVIN The neutral lipids were separated by T L C o n Silica Gel G plates using the solvent system 1.2-dichloroethane: acetic acid (100:I, v/v), and the radioactivity of each lipid species was measured. C P G and E P G were separated by preparative TLC, employing Silica Gel G plates. Plates were developed at 5°C in the solvent system chloroform: methanol :acetic acid: water (25: 15:3 :2, by et a / . (1964). Areas containing vol.) as described by SKIPSKY the labelled lipids were identified by comparison with standards. The tissue phospholipids were covered with a glass plate while the markers were briefly exposed to iodine vapours. Areas corresponding to C P G and EPG spots were scraped off the silica plates and placed in L cm dia. sintered glass columns. Diethyl ether (10 ml) was added to remove any acetic acid or other impurities. followed by methanol (20 ml) to elute the phospholipids. Recovery of radioactivity was in excess of 93 per cent. Rechromatography in chloroform:methanol-7 N ammonia (65:35:5) revealed that the E P G and C P G isolated by this method were free of radioactive contaminants. Gas-liquid chroniutoymphy. Gas chromatography of the fatty acid methyl esters was performed on a Barber ~ C o l m a n gas chromatograph equipped with a Hewlett-Packard radioactivity monitor. The stationary phasc of the column was 15% DEGS o n Chromosorb WAW (Supelco, Bellefonte, PA). The column was operated at 200°C under 25 Ib argon pressure. Peak areas were quantified by a HewlettPackard integrator Model 3370B or by triangulation. The various intermediates in the course of polyenoic fatty acid formation were identified by methods already described in detail (YAWN& MENKES.1974).

Polyunsaturated fatty acid metabolism

73

SPG was significantly less than in control cultures, or in cultures treated with dBcAMP or subjected to The morphology of neuroblastoma cells under a variety afenvironmental conditions is depicted in Fig. removal of serum (Table 1). By contrast, incorporation 1. Cells in the logarithmic phase have epithelioid-like or radioactivity into triacylglycerols by cytosine araappearance and possess only a few, small processes binoside-treated cultures was twelve times that of con(Fig. la). Cells in the stationary phase, induced by trol cultures, and was greater than under the other enaddition of cytosine arabinoside, an inhibitor a DNA- vironmental changes studied. The uptake of [1-14C]linolenic acid into various synthesis (BYFIELD & KARLSSON, 1973)(Fig. lc), or by removal of serum (Fig. Id), exhibit long neuritic exten- cellular lipids is depicted in Table 2. The distribution sions. Contrary to observations of PRASAD & HSIE of radioactivity between neutral and polar lipids (1971).FURMANSKI et al. (1971) and LIM& MITSUNOBUresembled that obtained for linoleic acid. There were, (1972),who studied other mouse C1300 neuroblastoma however, some differences between incorporation patclones, addition of dBcAMP, in concentrations up to terns. When linolenic acid was used as a precursor, 57 1 mM, failed to increase the length of cell processes to per cent of total cellular radioactivity had, by the end any significant extent by the time when metabolic of 48 h, entered the EPG fraction. The ratio of radioactivity between CPG and EPG fractions obtained with studies were initiated (Fig. lb). linolenic acid was lower than was obtained with linoleic acid. Differential counts between phosphatidal Distribution of radioactivity between lipid classes ethanolamine (plasmalogen) and phosphatidyl ethaWhen neuroblastoma cells were pretreated for 48 h nolamine showed that the proportion of radioactivity with either cytosine arabinoside, serum-free medium, or dBcAMP, and incubated with [l-'4C]linoleic acid, in plasmalogen was greater when linolenic rather than 8C95percentofthe radioactivity was incorporated into linoleic acid was used as precursor (Table 3). With the phospholipid fraction. After 24 or 48 h incubation both substrates the proportion of radioactivity enterwith labelled linoleic acid, CPG, EPG and DPG were ing plasmalogens increased when cells were pretreated labelled preferentially (Fig. 2; Table 1). Removal of with cytosine arabinoside or by removal of serum. While a significant proportion of labelled linoleic acid serum from the medium or treatment with dBcAMP entered DPG, this fraction remained unlabelled when did not affect total incorporation of radioactivity (as linolenic acid was used as precursor. measured in d.p.m./pg DNA) or distribution of RESULTS

radioactivity between the various phospholipids. In cells pretreated with cytosine arabinoside, total incorporation of isotope was reduced 25 per cent. In these cultures the amount of radioactivity in EPG, DPG and TABLE1. DISTRIBUTION OF RADIOACTIVITY AMONG THE MAIN

Lipid class Triacylglycerol Cholesterol ester Free fatty acid CPG SPG* EPG DPG Total incorporationt

Elongation and desaturation of essential fatty acids After 48 h incubation with [1-14C]linoleic acid, 26 per cent of the incorporated fatty acid was converted LIPID CLASSES AFTER INCUBATION WITH [1-'4C]LINOLEIC

Control

Complete medium fdBcAMP (1 x 1 0 - 3 ~ )

Cyt. arab. (2 x mM)

16f3 0 30 _+ 6 1082 f 133 356 f 41 592 _+ 103 617 f 72

40 3 46 1146 304 75 1 536

195 f 31 39 f 8 72 f 14 1112 f 144 204 f 62 290 f 43 126 f 58

106 9 56 1429 408 590 443

2780

2900

2070

3120

ACID

+

!

Serum-free medium

* Also contains Inositol-PG. t Incorporation into other, minor fractions is not included in this table. Details of the experimental procedure are described in Methods. Cells in the logarithmic phase were plated in 60 mm petri dishes and allowed to grow for a period of 48 h. Then the medium was removed and complete medium (10% fetal calf serum) with either dBcAMP or cytosine arabinoside (cyt. arab.) were added. A fourth group of cells was incubated with serum free medium. After 48 h at 37°C the preincubation medium was replaced by the same corresponding medium to which [ l-'4C]Iinoleic acid (0.4 x lo6 d.p.rn./petri dish) was added, and after 48 h incubation a t 37°C cells were harvested and cellular lipids were extracted and isolated. Values are expressed as d.p.m./pg DNA and represent the mean +s.D. of three petri dishes.

74

E. Y A V I N , 2.YAWNand J. H. MENKES

TABLE 2. DISTRIBUTION OF RADIOACTIVITY AMONG THE MAIN LIPID CLASSES IN NEUROBLASTOMA CELLS AFTER [ ~-'4c]LINOLENIC ACID

Lipid class Triacylglycerol Cholesterol ester Free fatty acid CPG SPG* EPG Total incorpora tion

Control

Complete medium +dBcAMP (1 x 1 0 - 3 ~ )

73 f 6 20 3 30 3 634 f 60 611 f 76 1899 f 100

*

12 3 15 574 475 1937

3320

3090

+ Cyt. arab. (2 x mM) 172 k 18

*

10+3

34 5 664 k 68 287 t 49 1346 i: 78 261 5

INCUBATION WITH

Serum-free medium

25

3 3*3 13 3 530 34 433 ri. 31 1934 64

*

3140

Experimental conditions are the same as described for Table 1 except that [l-'4C]linolenic acid (0.5 x lo6 d.p.m./petri dish) was added. Values expressed as d.p.m./pg DNA represent the mean S.D. of three petri dishes. For abbreviations see Table I . * Also contains Inositol-PG.

TABLE3. PHOSPHATIDAL/PHOSPHATIDYL ETHANOLAMINE

RATIO AFTER INCUBATION OF NEUROBLASTOMA CELLS WITH '4c]LINOLEIC AND [ 1 -14C]LINOLENIC ACIDS

Fatty acid precursor Linoleic acid Linolenic acid

Control 066 2.09

Complete medium (phosphatidal/phosphatidyl ratio) +dBcAMP + Cyt. arab. ( I x 10-3 M) (2 x mM) 0.73 2.04

1.50 2.58

[ 1-

Serum-free medium I .07 2.21

Phosphatidal (I-alkenyl, 2-acyl) and phosphatidyl (I-acyl. 2-acyl) ethanolamine phosphoglycerides were isolated by two dimensional TLC and counted as described in Methods. Experimental conditions were the same as described for Tables 1 and 2.

to 20 :3 ru6 and 20 : 4w6 polyunsaturated fatty acids (Table 4).The time course for linoleic and eicosatrienoic acid (20 :3) incorporation into EPG and CPG of cellular lipids growing in the logarithmic phase is shown in Fig. 3a. It is evident that the extensive labelling of the CPG fraction (Table 1) is almost entirely due to incorporation of linoleic acid. The CPG pool becomes rapidly labelled, reaches a plateau and then

decreases continuously over the 24-h period studied. Similar experiments, performed with [l-'4C]linolenic acid, showed that most of the radioactivity accumulated in the eicosapentaenoic acid (20:5w3) species. (Fig. 3 b). The rate of 20 : 50.13 formation was rapid, and between 1 and 4 h incubation it was the major labelled fatty acid. As shown in Fig. 3b, most of the 20 : 5w3 was incorporated into EPG. After 8 h incubation, incor-

TABLE 4. DISTRIBUTION OF RADIOACTIVITY IN FATTY ACIDS OF THE POLAR LIPIDSOF NEUROBLASTOMA AFTER INCUBATIONWITH [ 1-14c]LINOLEIC ACID

Fatty acid 18:2u6 20 :2~06 20 : 306 20 : 4u6 22 : 4w6* l6:O-IX:I

Control

Complete medium + dBcAMP ( I x 1 0 - 3 M)

+ Cyt. arab. (2 x mM)

Serum-free medium

72.9 0.9 13.6 12.3 0 0.2

80.8 2.0 11.4 5.2 0 0.3

69.5 1.6 11.5 14.2 2.1 1 .o

77.6 2.3 11.9 7.9 0 0.2

Experimental conditions are the same as described for Table 1. Fatty acids of the polar lipids were subjected to methanolysis and the methyl esters analyzed by GLC as described in Methods. Values are expressed as per cent distribution and represent pooled lipid extracts from three to four petri dishes. * Identified by retention time.

FIG. 1. Morphology of N-18 cells under various experimental conditions. Phase contrast pictures taken at the end of incubation with the radioactive fatty acids, 5 days after plating. Cells in log phase under control conditions (a); 1 mM dBcAMP (b); +2 x 1 O - j mM cytosine arabinoside (c) and in serum-free medium (d). For experimental details see Methods. The bar represents 100 pm.

+

NC-74

.

-_

- ..-

-

.-

- -. -_- --

-

FIG.2. Radioscanning of the lipid classes, separated by two-dimensional TLC after incubation with [l-14C]linoleicacid. Experimental conditions were the same as described for Table 1, except that incubation was carried out for 24 h. For details see Methods. The lipid classes marked numerically are as follows: 0, Origin; 1, inositol-PG; 2, SPG; 3, CPG; 4, lyso Ethanolamine-PG (from plasrnaIogen); 5, EPG; 6, DPG; 7, glycolipids, 8, TG. A-Cytosine arabinoside treated cells; B-dBcAMP treated cells; C-serum-free treated cells; D-control cells.

Polyunsaturated fatty acid metabolism

15

Although 20:2w6 and 20:306 were detected, we were unable to establish with certainty in the present study whether 18 :306 or 20:2w6 was the first intermediate in the formation of arachidonic acid (20 : b 6 ) from linoleic acid by neuroblastorna cells. A n unidentified radioactive peak, tentatively identified as 18 : 306 on the basis of GLC retention times, was detected whenever cells were incubated for short periods. Only traces of this peak could be detected when cultured dissociated brain cells were incubated under analogous conditions. This compound was not found in the medium, and was not an impurity of the labelled substrate. The distribution of label between the various polyunsaturated fatty acids of the linolenic acid series is shown in Table 5. More than 90 per cent of labelled INCUBATION TIME (h) linolenic acid was converted into other polyunsaturated fatty acids, 20 :503 being the most unsaturated 1400 acid detected. Our failure to find significant amounts of radioactivity in 20 :303 suggests that this compound either has a rapid turnover or is'not a n intermediate in the conversion of linolenic acid to higher polyunsaturated fatty acids of the w3 series. With shorter incubation periods, we detected an unidentified radioactive peak, tentatively considered to be 18 :403 on the basis of its GLC retention time. This compound could not be demonstrated when 14-day-old cultured dissociated brain cells were incubated with labelled linolenic acid under analogous conditions. When neuroblastoma cells were incubated with either linoleic or linolenic acids only minimal amounts INCUBATION TIME ( h ) of radioactivity entered palmitic and stearic acid, indiFIG. 3. Incorporation of major radioactive fatty acids into cating that there was little recycling of two-carbon the choline and ethanolamine phosphoglycerides classes. fragments obtained by p-oxidation of polyunsaturated Cells grown in complete medium for 48 h were incubated for fatty acids (Tables 4 and 5). There was no significant various periods of time with [1-'4C]linoleic acid (Fig. 3a), and [ 1-'4C]linolenic acid (Fig. 3b). Conditions were those difference in the distribution of label when incubation described in Tables I and 2. The CPG and EPG classes were conditions were altered by addition of dBc AMP, cytoisolated, and fatty acid content was analysed by gas chro- sine arabinoside, or by withdrawal of serum from the matography as described in Methods. Values are expressed medium (Tables 4 and 5). as d.p.m./pgDNA and represent pooled lipid extracts of Figure 4 shows the distribution of label between three 60 mm petri dishes. I8:2-Linoleic acid; 20:3--eico- fatty acids of cellular lipids extracted from neuroblassatrienoic acid. toma, cultured dissociated embryonic rat brain cells and astrocytoma after incubation with [1-'4C]linoleic poration of label into the 18:303 species of the EPG and [ I-'4C]linolenic acids. It is evident that the culfraction leveled off, and incorporation into the 18 :303 tured dissociated cells are better able to synthesize species of CPG began to diminish. An analogous 20:b6, 22:5w3, and 22:6w3 than the two types of phenomenon was observed for all polyunsaturated tumours. More than 60 per Cent of labelled linoleic fatty acid intermediates, and could be correlated with acid was converted to arachidonic acid by brain cell the low CPG/EPG labelling ratio obtained at 48 h in- cultures, while under analogous experimental conditions only 15 and 25 per cent were converted by neurocubation (Table 2). Analysis of the distribution of linoleic acid-derived blastoma and astrocytoma cells. In both tumour culradioactivity between the fatty acids of polar lipids un- tures labelled 22: 6w3 was barely detectable after 48-h der various incubation conditions is depicted in Table 4. incubation with radioactive linolenic acid while in

76

E. YAVIN,Z. YAVINand J. H. MENKES

TABLE 5. DISTRIBUTIONOF RADlOACTlVlTY

IN FATTY ACIDS OF THE POLAR 1.IPII)S OF NEUROBLASTOMA AFTER INCUBATION WITH [ ~-'4c]LINOLENIC ACID

Complete medium + dBcAMP Fatty acid 18:3w3 20 : 3 0 3

20 : 4w3 20 :503 22 : 5w3 16:0-18: I

Control

(1 x 10-3 M)

4.0 0 11.9 71.4 11.9 0.7

3.0 0 10.6 71.3

+ Cyt. arab. (2 x mM) 9.8 0

5.5 76.8 7.8 0

13.4 0.4

Serum-free medium 2.8 0.5 6.0 79.4 10.0 0.4

Experimental conditions are the same as described for Table 2. Fatty acid analysis was performed as described in Methods. Values represent per cent distribution of pooled lipid extracts from three to four petri dishes.

brain cell cultures labelled 22:6w3 was already detectable after 3-4 h incubation. By 48 h 22:6w3 contained some 30 per cent of total labelled fatty acids. It was also evident that the longer the brain cell cultures had been maintained in uitro, the less well they were able to synthesize 20:406 and 22:603.

DISCUSSION

The elongation and desaturation of essential fatty acids by mammalian cell cultures has been studied in a number of laboratories (MEAD& HAGGERTY, 1969: BAILEY r t al, 1972; STOFFEL & SCHEID, 1967). The main purpose of the present paper was to examine the metabolic patterns of essential fatty acids in tumour cells and to document any alterations that might accompany cellular differentiation and neuritic outgrowth. It is in the latter phenomenon that essential fatty acids may play a particularly important role.

Neuroblastoma cell cultures take up linoleic and linolenic acids and esterify them at approximately the same rate (Table 1 and 2). Between 80 and 95 per cent of total counts, representing 5C70 per cent of the administered dose, are incorporated into cellular phospholipids after 48 h incubation. The distribution of label between the phospholipid fractions differs for the two essential fatty acids employed as precursors. When h o l e i c acid was the precursor, CPG was the major labelled lipid, while with linolenic acid as precursor the EPG fraction was labelled most extensively. Forty per cent of total cellular radioactivity derived from [li4C]linoleic acid was found in CPG, while only some 20 per cent was esterified to C P G when [ I-i4C]linolenicacid was used as substrate. Most of the radioactivity derived from the latter entered the EPG fraction. The marked difference between the labelling pattern of CPG and EPG suggests that a direct transfer of 14Clabelled fatty acids from CPG to E P G is not a major

m Aslrccytomo =Rat Broin-6Div

2

50

25

FIG 4 Distribution of radioactivity In the fatty acids of total lipids in various cell cultures N-18 neuroblastoma, C6-astrocytorna. cultured dissociated rat cerebrum, 6 div and 18 div, were incubated for a period of 24 h with [1-'4C]l~noleic acid (Fig. 4a) and [1-'4C]linolenic acid (Fig. 4b) under similar conditibns as described for Tables 1 and 2 Aliquots of total lipids were treated with methanolic-HC1, and the resulting fatty acid methyl esters were analysed by GLC Values represent mean SD. of 3-4 cultures

Polyunsaturated fatty acid metabolism

pathway in neuroblastoma cultures. When cell division ceases, as is the case upon addition of cytosine arabinoside to the cultures, the patterns of fatty acid incorporation change. The diminished entry of label into EPG and SPG in the presence of cytosine arabinoside suggests that thc turnovcr of thcsc lipids is reduced when the cells are in the stationary phase. Treatment of cells with dBcAMP or by withdrawal of serum failed to alter the extent of fatty acid incorporation and its distribution among the various lipid fractions. The diminution of radioactivity in 20 :406 in the presence of dBc AMP (Table 4) suggests that the compound may directly affect the formation of the higher polyunsaturated fatty acids of the 0 6 series. Further support for this hypothesis is offered by the reduced levels of radioactivity in 20 :406 in cells grown in serum free medium, conditions known to increase the levels of cyclic A M P (PRASAD et al., 1973). Within the EPG fraction, phosphatidal ethanolamine (plasmalogen) is the major acceptor for the higher polyunsaturated fatty acids derived from linolenic acid (Table 3). When cell division is slowed down or arrested by removal of serum or addition of cytosine arabinoside, the relative amount of labelled polyunsaturated fatty acids in plasmalogens is increased. This suggests that plasmalogens may be intimately involved in cellular differentiation and in the formation of new cell processes. Our observations that normal brain cells may lose the ability to convert linoleic acid to arachidonic acid after an extended period in uitro (Fig. 4) parallels data by HARARY et a!. (1967) on beating heart cells cultures. The relationship of decreased enzymatic abilities to in uitro senescence is under investigation.

77

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SANTEN R. J. & AGRANOFF3. W. (1963) Biochim. biophys. Acta 73, 251-262. SEEDS N. W., GILMAN A. G., AMANOT. & NIRENBERG M. W. (1970) Proc. natn. Acad. Sci., U.S.A.66, 160-167. V. P., PETERSON R. F. & BARCLAY M. (1964) BioSKIPSKI chem. J. 90,374-378. STEINA. A., OPALKA E. & ROSENBLUM I. (1965) Cancer Res. 25,201-205. STOFFEL W. & SCHEIDA. (1967) 2.physl. Chem. 348, 205/ 206. S., ALLANA. & MILLINGTONR. H. (1953) Acknowledgernents--This work was supported by N. I. H. WEINHOUSE Cancer Res. 13, 367-371. Grants CA 13538 and NS 06938, and a grant from ChilYAWN E. & MENKESJ. H. (1973~)J. Cell. Biol. 57, 232-237. dren’s Brain Diseases, Inc. The authors wish to express their gratitude to Mrs. NATA- YAVINE. & MENKESJ. H. (1973b) J . Neurochem. 21, 901912. LIE STEIN and Mr. DONALD HARRIS for their technical assistance. YAVINE. & MENKES J. H. (1974) J. Lipid Res. 15, 152-157.