573

Incorporation of Arachidonic Acid and Eicosapentaenoic Acid into Phospholipids by Polymorphonuclear Leukocytes in vitro JEN-SIE TOU, Department of Biochemistry, Tulane University School of Medicine, New Orleans, LA 70112

ABSTRACT

The present study demonstrated that the patterns of the incorporation of [1J4C] arachidonic acid and [1J4C]eicosapentaenoic acid into individual phospholipids by polymorphonuclear leukocytes w e r e similar. However, human leukocytes exhibited higher activity than guinea pig peritoneal leukocytes in the formation of arachidonoyl- and eicosapentaenoyl-phosphatidic acid. Cells from both origins showed a decrease of label in phosphatidylcholine accompanied by an increase of label in phosphatidylethanolamine after a longer period (30-120 rain) of incubation, suggesting that part of the arachidonoyl or eicosapentaenoyl moiety in phosphatidylethanolamine may be derived from that of phosphatidylcholine. The observed difference between human cells and elicited cells in the timecourse of the incorporation of both fatty acids into phosphatidylcholine and phosphatidylethanolamine appears to be due to different contents of the diacyl and ether-linked class compositions of these phospholipids in cells from different origins. Both labeled fatty acids were incorporated more rapidly into the diacyl-linked class, but were retained to a greater extent in alkylacyl-phosphatidylcholine and alkenylacyl-phosphatidylethanolamine. The data suggest that, in addition to alkylacylphosphatidylcholine and phosphatidylinositol, alkenylacyl-phosphatidylethanolamine may be an important endogenous source of arachidonic acid and eicosapentaenoic acid in stimulated human leukocytes. Lipids 19:573-577, 1984.

Eicosapentaenoic acid, 20:5(n-3), is a major unsaturated component of fish lipid. It has been shown to be a little more active than arachidonic acid, 20:4(n-6), toward 5-1ipoxygenase in guinea pig peritoneal polymorphonuclear leukocytes (PMNS) (1). It is metabolized to 5,12-dihydroxyeicosatetraenoic acid or leukotriene Bs (LTBs) by these cells in response to ionophore A23187, and LTBs exhibits 10- to 30-fold less chemotactic potency for human PMNS than 5,12-dihydroxyeicosatetraenoic acid or leukotriene B4 (LTB4) (2). However, our knowledge is lacking about the esterification of exogenous eicosapentaenoic acid to phospholipids by PMNS. The present study was undertaken to compare the incorporation of exogenous [1J4C]20:4(n-6) into phospholipids with that of [ 1Jr 20:5(n-3) by intact PMNS in vitro. Although studies on the metabolism of arachidonic acid by PMNS in vitro have been carried out with cells either from human blood or from animal peritoneal exudates, elicited PMNS differ from human blood PMNS in a number of respects (3,4). In the present study, PMNS from human blood and from guinea pig peritoneal exudates were compared in their incorporation of radiolabeled arachidonic acid [20:4(n-6)] and eicosapentaenoic acid [20:5(n-3)] into phospholipids.

MATERIALS AND METHODS Preparation of PMNS

Human venous blood was collected from normal donors using heparin (1000 Units/ 50 ml blood) as an anticoagulant. The heparinized blood was diluted with an equal volume of Hanks balanced salt solution. In a 50 ml centrifuge tube the diluted heparinized blood was layered over one-third volume of Lymphocyte Separation Medium (Bionetics). After centrifuging the tubes at 400 x g for 30 min, PMNS and erythrocytes were found in the lower cell layer. After sedimentation of erythrocytes with two volumes of 3% dextran T500 (Pharmacia) and hypotonic lysis of contaminating erythrocytes, PMNS were washed and resuspended in Krebs-Ringer phosphate buffer modified to contain 1.3 mM CaC12 and 5 mM glucose at a concentration of 20 x 106 cells/ml. Cell counts were made in a hemocytometer, and cell viability was measured by trypan blue exclusion test. Cell preparations contained more than 95% PMNS. Guinea pig peritoneal PMNS were prepared as described previously (5). PMNS were collected 14 hrs after intraperitoneal injection of 12% sodium caseinate solution to guinea pigs. LIPIDS, VOL. 19, NO. 8 (1984)

J.-S. TOU

574

[1 j 4 C] Arachidonic acid (52.9 Ci/mol, New England Nuclear Corp.) or [1-14C]eicosapentaenoic acid (55.5 Ci/mol, New England Nuclear Corp.) was dissolved in dimethylsulfoxide (DMSO) and mixed with fatty acid-free bovine serum albumin (Mile Laboratory, 4 mg/ml 0.9% NaCt). The tubes, each containing 2.54 x l0 s dpm of [1J4C]arachidonic acid or 2.37 x l 0 s dpm of [ 1-14C] eicosapentaenoic acid, were preincubated at 37 C for 30 min and then mixed with 20 • 106 PMNS from human blood or from guinea pig peritoneal exudates in a final volume of 2 ml and further incubated. The final concentration of bovine serum albumin in the incubation medium was 0.1 mg/ml. The DMSO, whose final concentration in the incubation medium was 0.025%, had no adverse effect on cell viability. Incubations were performed at 37 ~ and stopped by the addition of 10 ml methanol to each tube.

Guinea pig peritonealPMNS

r

Incubation of Cells with Radiolabeled Fatty Acid

If')

2r,~

o [T-14C]20:4(~-6)

)

o

5

x

11

Lipid Extraction and Analysis

Lipids were extracted according to Bligh and Dyer (6). Phospholipids were resolved by twodimensional thin-layer chromatography on silica gel H (Analtech) and analyzed as described previously (7). The chromatogram was developed with chloroform-methanol-28% aqueous ammonia (65:25:5, v / v ) i n the first dimension and then with chloroform-methanol-acetic acid-water (85:10:40:4.5, v/v) in the second dimension. PC and PE purified according to (8) were each treated with B a c i l l u s c e r e u s phospholipase C (Sigma) and followed by acetylation of the diglycerides (9). The resultant 1-radyl-2-acyl-3acetylglyeerols were resolved into 1-alkenyl-2acyl-3-acetylglycerol, 1-alkyl-2-acyl-3-acetylglycerol and 1,2-diacyl-3-acetylglycerol by thinlayer chromatography (TLC) according to (10) on silica gel H (Analtech). The radioactivity of each lipid class was measured by liquid scintillation. RESULTS A N D DISCUSSION

The present study demonstrated that the patterns of the incorporation of 20:4(n-6) and 20:5(n-3) into individual phospholipids by PMNS were similar. It is likely that 20:4(n-6) in PMNS will be replaced by 20:5(n-3) when animals are fed a diet containing fish lipid. The lower chemotactic potency of LTBs derived from 20:5(n-3) than that of LTB4 derived from 20:4(n-6) (2) will consequently alleviate the process of inflammation. Figure 1 shows the similarity of the timecourse of the incorporation of [ 1j 4 C ] 20:4(n-6) LIPIDS, VOL. 19, NO. 8 (1984)

0

10

20

30

40

60

120

Minutes FIG. 1. The incorporation of [ 1J* C] 20:4(n-6) and [1J4C] 20:5(n-3) into PA by PMNS as a function of time. PMNS (20 • 106 ceils) from human blood or from guinea pig peritoneal exudates were incubated at 37 C at the indicated time with [1J4C] 20:4(n-6) (2.54 X 10~ dpm) or [1J4C]20:5(n-3) (2.37 X 105 dpm) as described in Materials and Methods. Each point represents the average value from three experiments. and [ 1 J 4 C ] 2 0 : 5 ( n - 3 ) into phosphatidic acid (PA) by PMNS from human blood and from guinea pig peritoneal exudates. However, human PMNS exhibited higher activity than guinea pig peritoneal PMNS in the formation of [1-14C]20:4(n-6)- and [1-1acI20:5(n-3)-PA. The labeled PA may participate in the biosynthesis of other phospholipids and neutral glycerolipids. The labeling patterns of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by both fatty acids in human PMNS were different from those in guinea pig peritoneal PMNS. In human PMNS the radioactivity of PC reached its maximum after 30 rain incubation and started to decline, whereas that of PE and phosphatidylinositol (PI) continued to increase (Fig. 2). In guinea pig peritoneal PMNS (Fig. 3), the radioactivity of PC reached its maximum between 20 and 30 min; at 120 min it was approximately 60% of that at 20 min. During the incubation period the phosphorus content of each phospholipid remained unchanged. The observed decline in the radioactivity of PC appears to be the consequence of an exhaustion

2o:4(n-6) and 20:5(n-3)in PMN Phospholipids

575 PE

12

~

-~

8

b x

Pl

"

c

~

2 , __ 0

20

40

60

80

, 100

PS ~ 120

M,nutes

FIG. 2. The incorporation of [1-~4CI20:4(n-6) into PC, PE and PI by human PMNS as a function of time. Incubation conditions and phospholipid analysis were described in Materials and Methods. Each point represents the average value -+ S.D. from three experiments. of labeled arachidonic acid and a rapid metabolism of the arachidonoyl moiety in PC, since it could be restored by a further addition of labels. The radioactivity of PE, on the other hand, was increased with incubation time, and it became higher than that of PC after 30 min incubation. The results suggest that part of the 20:4(n-6) moiety in PE may be derived from 20:4(n-6)-PC. The time-course of [1-14C]20:5 (n~3) incorporation into phospholipids by PMNS from both sources resembled that of [ 1-14C 120:4(n-6) (data not shown). Since quantitative analyses of the diacyllinked and ether-linked classes of PC and PE in PMNS from human blood and guinea pig peritoneal exudates have been reported (11,12), in the present study only the distribution of [ 1-14C120:4(n-6) and that of [ 1-14C] 20:5(n-3) among these class compositions were measured. Figure 4 shows the chromatographic resolution of alkenylacyl-, alkylacyl-, and diacyl-glyceride acetates derived from PC and PE. PC and PE from human PMNS contain more ether-linked class than diacyl-linked class, whereas the reverse is true for those from guinea pig peritoneal PMNS. It remains to prove whether the low content of ether linked PC and PE in guinea pig peritoneal PMNS is a characteristic of elicited PMNS or a reflection of the lipid pattern of guinea pig blood PMNS. It is noted

~F..... 0

~_ , 20

40

i

,

,

60

80

I00

120

N~lnutes

FIG. 3. The incorporation of [l-t*C]20:4(n-6) into PC, PE and PI by guinea pig peritoneal PMNS as a function of time. Incubation conditions and phospholipid analysis were described in Materials and Methods. Each point represents the average value • S.D. from three experiments. that the content of alkenylacyl-PE in guinea pig peritoneal PMNS obtained in the present study is lower than that obtained by Sugiura et al. (12). Further study is required to prove whether the discrepancy is a result of the use of different time periods in collecting PMNS from guinea pig peritoneal exudates. In the present study PMNS were collected 14 hrs after intraperitoneal injection of 12% sodium caseinate solution to guinea pigs; the time period of elicitation was 2 to 4 hrs longer than that performed by Sugiura et al. (12). If ethanolamine plasmalogen is one of the endogenous sources of 20:4(n-6) in stimulated PMNS, the content of this class of PE is expected to be decreased after a longer period of elicitation. As demonstrated in Table 1, the rate of esterification of [ 1 - 1 4 C ] 2 0 : 4 ( n - 6 ) i n t o diacyllinked and ether-linked PC and PE differed. The arachidonoyl moiety in diacyl-linked PC and PE appears to be more metabolically active than the corresponding ether-linked class. It was more rapidly incorporated into the diacyllinked PC and PE by PMNS from both sources, but it was retained to a greater extent in alkylacyl-PC and alkenylacyl-PE. After 2 hr incubation of human PMNS with [1-14C]20:4 (n-6) the radioactivity in diacyl-PC and alkylacyl-PC became comparable and that in alkenylLIPIDS, VOL. 19, NO. 8 (1984)

576

J.-S. TOU TABLE 1 D i s t r i b u t i o n o f [ ~4C] A r a c h i d o n i c A c i d in Diacyl-, A l k y l a c y l and Alkenylacyl-phosphatidyicholine and -phosphatidylethanolamine* Human PMNS Phosphatidyleholine

Time (Min) 10 20 30 60 120

Diacyl 83.1 72.5 67.7 60.3 54.1

Phosphatidylethanolamine

Alkylacyl (%)

Alkenylacyl

16.0 25.3 29.9 37.1 43.3

0.90 2.21 2.40 2.60 2.60

D i a c y | Alkylacyl (%) 62.2 48.6 38.9 33.3 26.3

12.6 15.4 14.5 13.3 13.0

Alkenylacyl 25.2 36.0 46.6 53.4 60.7

Guinea Pig Peritoneal PMNS Phosphatidylcholine Time (Min) 10 20 30 60 120

Diacyl 92.5 89.4 86.2 84.1 82.5

Phosphatidylethanolamine

Alkylacyl (%)

Alkenylacyl

6.70 9.75 12.6 14.4 15.8

0.80 0.85 1.20 1.50 1.70

D i a c y l Alkylacyl (%) 93.1 89.3 84.7 83.1 83.0

3.66 5.24 8.13 7.30 6.50

Alkenylacyl 3.24 5.46 7.17 9.60 10.5

* P M N S ( 2 0 X 106 cells) f r o m h u m a n b l o o d a n d g u i n e a pig p e r i t o n e a l e x u d a t e s w e r e s e p a r a t e l y i n c u b a t e d w i t h [ 1 A 6 C ] 2 0 : 4 ( n - 6 ) ( 2 . 5 4 X 106 d p m ) at 3 7 C a t i n d i c a t e d t i m e . PC a n d PE w e r e p u r i f i e d a n d t r e a t e d w i t h p h o s p h o l i p a s e C as d e s c r i b e d in Materials a n d M e t h o d s . The a m o u n t o f r a d i o a c t i v i t y in e a c h class o f t h e r e s u l t a n t d i g l y c e r i d e a c e t a t e s is t h e a v e r a g e value f r o m t w o e x p e r i m e n t s a n d is e x p r e s s e d as a p e r c e n t o f t h e t o t a l r a d i o a c t i v i t y r e c o v e r e d f r o m t h e t h i n - l a y e r plate. Atkenylacyl 9 9

9 9

PC PE From g u i n e a p i g p e r i t o n e a l t',~l S

9 9

A/kylacyl "

Diacyl

PC PE l'rorn Human PiiNg

FIG. 4. TLC separation of diglyceride acetates derived from PC (1.2 ug phosphorus) and PE (1.2 ug phosphorus) isolated from human PMNS or from guinea pig peritoneal exudates. The thin-layer plate of silica gel H was first (7 cm) developed with hexane: ether (1:1, v/v), then (15 cm) with toluene (10). Spots were detected by charting the plate with 50% sulfuric acid containg 0.06% potassium dichromate. acyl-PE comprised 61% of the total radioactivity in PE fraction. Under identical incubation conditions a small but significant amount of radioactivity was detected in alkenylacyl-PC and alkylacyl-PE. In guinea pig peritoneal PMNS, the percentage of radioactivity in alkylacyl-PC and in alkenylacyl-PE was much lower than that in human cells. The marked loss of label in PC from guinea pig peritoneal PMNS after a longer incubation period is probably accounted for by the higher contents of LIPIDS, VOL 19, NO. 8 (1984)

diacyl-linked PC in these cells than in human cells. A more rapid metabolism of the 20:4(n-6) group in the diacyl-PE than in the alkenylacylPE was also demonstrated in rat testis during essential fatty acid deficiency (13). Further studies are required to determine whether the transfer of arachidonoyl group from PC to PE is catalyzed by a coenzyme A dependent acyltransferase (14) or by a recently discovered transacylase in platelets [15). The retainment of [ 1 ) 4 C ] 2 0 : 4 ( n - 6 ) by alkylacyl-PC and alkenylacyl-PE to a greater extent in vitro than the corresponding diacyllinked class demonstrated in the present study is compatible with the distribution of endogenously esterified 2 0 : 4 ( n - 6 ) i n vivo (11,12). In human PMNS the enrichment of 20:4(n-6) in alkenylacyl-PE suggests that, in addition to PC and PI (16), ethanolamine plasmaiogen may be an important endogenous source of 20:4(n-6) in stimulated ceils for the synthesis of lipoxygenase products. Indeed, a loss of endogenous 20:4(n-6) from both PC and PE was identified in PMNS from human patients with chronic myelogenous leukemia after phagocytosis (I 7).

577

20:4(n-6) and 20:5(n-3) in PMN Phospholipids ACKNOWLEDGMENTS

8.

This work was supported by research grants from the American Heart Association-Louisiana, Inc., and from Cancer Crusaders, New Orleans, Louisiana. I wish to thank Drs. Toru Nishihara and Arthur Gotlieb for providing me human PMNS.

9. 10.

REFERENCES

11.

1. Ochi, K.; Yochimoto, T.; and Yamamoto, S. (1983) J. Biol. Chem. 258, 5754-5758. 2. Goldman, D.W.; Pickett, W.C.; and Goetzl, E.J. (1983) Blochem. Blophys. Res. Comm. 117, 282-288. 3. Takamori, K., and Yamashita, T. (1980) Inf. lmm. 29, 395-400. 4. Pember, S.O.; Barnes, K.C.; Brandt, S.J.; and Kinkade, Jr., J.M. (1983) Blood 61, 1105-1115. 5. Tou, J.-S. (1979) Blochim. Biophys. Acta 572, 307-313. 6. Bligh, E.C., and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-91"/. 7. Tou, J.-S. (1978) Biochim. Blophys. Acta 531, 167-178.

12. 13. 14. 15. 16. 17.

Tou, J.-S. (1981) Blochim. Blophys. Acta 665, 491-497. Waku, K.: Ito, H.; Blto, T.; and Nakazawa, Y. (1974)1. Biochem. 75, 1307-1312. Rendonen, O., and Luukkonen, A. ( 1 9 7 6 ) i n Lipid Chromatographic Analysis, Vol. 1, 2nd Edition (Marinetti, G.V, ed.), p. 37, Marcel Dekker, New York. Mueller, H.W.; O'l'laherty, J . T . ; a n d Wykle, R.L. (1983) Fed. Proc. 42, 2230. Sugiura, T.; Onuma, Y. ; Sekiguchi, N.; and Waku, K. (1982) Biochim. Biophys. Acta 712, 515-522. Blank, M.L.; Wykle, R.L.; and Snyder, F. (1973) Blochim. Biophys. Acta 316, 28-34. Trotter, J.; Flesch, I.; Schmidt, B.; and Ferber, E. (1982)J. Biol. Chem. 257, 1816-1823. Kramer, R.M., and Deykin, D. (1983) J. Biol. Chem. 258, 13806-13811. Walsh, C.E.; DeChatelet, L.R.; Chilton, F.H.; Wykle, R.L.; and Waite, B.M. (1983) Blochim. Blophys. Acta 750, 32-40. Smolen, I.E., and Shohet, S.B. (1974) J. Clin. lnves. 53, 726-734. [ R e v i s i o n r e c e i v e d F e b r u a r y 17, 1 9 8 4 ]

L1P1DS, VOL. 19, NO. 8 (1984)