The metabolism of (n − 3) and (n − 6) fatty acids and their oxygenation by platelet cyclooxygenase and lipoxygenase

Prog. Lip~l Res. Vol. 25, pp. 19-28, 1986 Printed in Great Britain. All rights reserved

0163-7827/86/S0.00 + 0.50 © 1986 Pergamon Journals Ltd

T H E M E T A B O L I S M O F ( n - 3) A N D ( n - 6) F A T T Y ACIDS AND THEIR OXYGENATION BY PLATELET CYCLOOXYGENASE AND LIPOXYGENASE HOWARD SPP~CI~R The Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio 43210, U.S.A.

INTRODUCTION The eicosanoids produced from (n - 3 ) and (n - 6 ) fatty acids mediate physiological processes in diverse ways. In platelets, arachidonic acid is metabolized primarily to thromboxane A2, HHT and 12-HETE.tS Thromboxane A2 is a potent stimulant of platelet aggregation and a vasoconstrictor. ~9 5,8,11,14,17-Eicosapentaenoic acid (EPA) is a relatively poor substrate for platelet cyclooxygenase but it is converted to small amounts of thromboxane A3.~5Thromboxane A3 is a poor agonist for inducing platelet aggregation. 3°,u In fact, simultaneous release of arachidonic acid and EPA will result in reduced thromboxane A2 synthesis due to competition of these two acids for cyclooxygenase.3°.44 In addition, 12-HPETE stimulates the synthesis of thromboxane A3.28 Collectively, these observations help explain the findings of Dyerberg and Bang 7 who showed that the prolonged bleeding time in Eskimoes is related to the high level of EPA in their membrane phospholipids. In the artery, arachidonic acid is metabolized primarily into prostacyclin-I,.21 It is a potent inhibitor of platelet aggregation and exerts its effects by elevating cAMP. 3.45 It relaxes arterial smooth muscle.m4When endoperoxides produced from EPA are incubated with arterial preparations, they are metabolized to prostacyclin-I3, which is also an inhibitor of platelet aggregation and relaxes arterial strips and smooth muscle)°'3s EPA itself is a rather poor substrate for prostacyclin synthesis, but its synthesism!like that of thromboxane A,) ° has recently been shown to take place in man. Peritoneal and circulating neutrophils metabolize arachidonic acid into a number of different leukotrienes. In lung, arachidonic acid is metabolized into a number of peptidoleukotrienes. The synthesis and biological properties of these compounds have recently been reviewed by Samuelsson. ~ EPA is also metabolized into a family of leukotrienes.2°~s~'~'40 Several studies show that leukotriene B5 is less potent than leukotriene B4 as a chemotactic agent and in inducing neutrophils to degranulate. 25.34,4° From the above introduction, it is apparent that interactions may exist between arachidonic acid and EPA and their metabolites to mediate a number of physiological processes. We thus carried out 3 different types of experiments to define what differences exist between ( n - 3) and ( n - 6) fatty acid metabolism. In the first study, rats were maintained on a diet in which the sole source of fat was either ethyl linoleate, ethyl linolenate or an equal mixture of these 2 ethyl esters. We then analyzed the fatty acids from individual platelet and liver phospholipids. The purpose of these studies was to define what differences exist in the overall metabolism of (n - 3) and (n - 6) fatty acids and their subsequent acylation into phospholipids. Neither human platelets31 nor megakaryocytes from the guinea-pig37 have a 6-desaturase, but megakaryocytes do have a 5-desaturase. 37 Presumably, platelets obtain their fatty acids at least in part by uptake from plasma, the composition of which in part is regulated by liver metabolism. Secondly, a number of e x v/vo studies were carried out to more precisely define what regulates the incorporation of long chain (n - 3 ) acids, as found in fish oil, '2 into human platelet phospholipids. Thirdly, platelet and other membrane phospholipids contain other (n - 3 ) and (n - 6 ) fatty acids. We thus defined how these acids were metabolized by platelet iipoxygenase and cyclooxygenase. 19

20

H.

METABOLISM

OF

(n-3)

AND

Sprecher

(n-6) POLYUNSATURATED IN THE RAT

FATTY

ACIDS

Male weanling Sprague--Dawley rats were maintained on a semisynthetic diet for 5 weeks in which the sole source of fat was either 3% by weight of ethyl linoleate, ethyl linolenate or 1.5% of each of these ethyl esters. Phospholipids from platelets and liver were separated by thin layer chromatography 9 and methyl esters were separated by gas liquid chromatography (GLC). The results in Tables 1-4 compare the amounts of (n - 3) and (n - 6 ) fatty acids found in individual platelet and liver phospholipids? 2 When linoleate was fed as the sole source of fat, there were no major differences in the amounts of arachidonic acid found in individual phospholipids vs. in chow fed controls. The only exception was liver PS where arachidonic acid increased from 22.7% in chow fed controls to 38.3% in those animals receiving linoleate. Linolenate itself was not incorporated to any significant extent into any phospholipid. There was, however, a marked increase in the amount of EPA incorporated into all phospholipids. Arachidonic acid and EPA are structurally similar. If these two acids are recognized to the same extent by all the enzymes

T~t~

Fatty Acid Composition in % of Rat Platelet and Liver Phosphatidylethanolamine After Feeding Different Ethyl Esters

1. (n - 6 ) a n d (n - 3)

Dietary fatty

acid

Platelets Fatty acid

Chow

18:2

18:3

18:2(n - 6)

3.8 46.8 7.8 --

1.5 53.7 10.4 --

1.3

-----

20:4(n -6) 2 2 : 4 ( n - 6) 2 2 : 5 ( n - 6) 18:3(n 20:5(n 22:5(n 22:6(n

-

3) 3) 3) 3)

--3.8 1.0

Liver 18:2/18:3

Chow

18:2

18:3

18:2/18:3

7.7 30.3 0.2 --

5.1

13.2 -0.5

4.0 37.7 3.4 --

35.8 0.9 5.8

1.3 4.5 -0.1

3.6 18.6 0. I 0.3

0.5 39.5 10.5 1.0

-9.3 4.8 2.1

-1.1 1.5 10.3

---2.4

0.7 19.3 3.5 17.6

-5.4 2.4 19.4

Fatty Acid Composition in % of Rat Platelet and Liver Phosphatidylcholine After Feeding Different Ethyl Esters

T A a t ~ 2. (n - - 6) a n d (n - 3)

Dietary fatty

acid

Platelets

Liver

Fatty acid

Chow

18:2

18:3

18:2/18:3

Chow

18:2

18:3

18:2/18:3

18:2(n 20:4(n 22:4(n 22:5(n

-

6) 6) 6) 6)

11.6 5.3 . .

7.0 8.7

3.2 1.1

8.8 3.1 .

18.2 24.5

11.3 25.6

9.7 12.6

0.3

2.7

3.6 3.8 0.2 --

18:3(n 20:5(n 22:5(n 22:6(n

-

3) 3) 3) 3)

-0.6 -.

-0.7 0.5 4.1

---0.9

0.8 18.7 1.6 7.6

0.2 5.5 1.1 9.0

.

.

.

.

.

1.4 4.4 0.5 .

---.

.

.

.

-1.0 --

0.1

Fatty Acid Composition in % of Rat Platelet and Liver Phosphatidylserine After Feeding Different Ethyl Esters

TASTE 3. (n - - 6) a n d (n - 3)

Dietary fatty

acid

Platelets Fatty acid

Chow

18:2(n 20:4(n 22:4(n 2 2 : 5(n

--

6) 6) 6) 6)

6.7 33.5 2.1 .

18:3(n 20:5(n 22:5(!1 22:6(n

----

3) 3) 3) 6)

. -1.8 0.6

18:2 2.6 32.5 2.4 .

. .

18:2/18:3

Chow

4.5 11.5 1.9 .

4.7 23.9 1.5

2.1 22.7 -0.9 0.5 1.0 13.6

. ----

Liver

18:3

. 10.2 7.5 --

.

18:2

18:3

18:2/18:3

10.2 38.3 0.5 5. I

3.0 3.2 ---

8.2 19.6 -0.8

--1.0

0.6 12.4 2.7 6.1

5.9 1.7 I 1.4

. 1.1 2.1 --

Metabolism o f fatty acids

21

TABLE 4. (n -- 6) and (n - 3) Fatty Acid Composition in % o f Rat Platelet and Liver Phosphatidylinositol After Feeding Different Ethyl Esters Dietary fatty acid Platelets Fatty acid

Chow

18:2(n 20:4(n 22:4(n 22:5(n

-

6) 6) 6) 6)

1.4 40.3 1.0 .

18:3(n 20:5(n 22:5(n 22:6(n

-

3) 3) 3) 3)

. --.

Liver

18:2

18:3

18:2/18:3

Chow

18:2

18:3

18:2/18:3

0.7 39.7 0.9 . .

1.1 14.9 -.

0.9 29.0 --

2.0 39.7 0.3 0.4

1.3 39.9 0.4 3.1

0.4 13.8 -0.2

1.2 30.0 0.3 0.3

. --0.3

4.8 11.7 9.5

1.0 3.5 5.6

.

. ---

.

.

.

. 2.2 1.7

9.4 3.6 .

.

. -1.0 1.9

TABLE 5. Ratio o f 20:5(n--3) to 2 0 : 4 ( n - 6 ) in Platelet and Liver Phospholipids after Feeding Diets Containing Ethyl Linolenate or Ethyl Linoleate plus Ethyl Linolenate PE

PC

PS

Pl

Platelets 18:3 diet 18:2/18:3 diet

3.0 0.2

4.0 0.3

0.9 0.04

0.6 0.07

Liver 18:3 diet 18:2/18:3 diet

4.3 0.3

4.9 0.4

3.8 0.3

0.4 0.03

T~SLE 6. Reaction Rates for Desaturation and Chain Elongation o f (n - 6 ) and (n - 3 ) Fatty Acid using Rat Liver Microsomes Microsomal protein (nmois/min/m8)

(n -

6)

Sequence

9,12-18:2.--~6,9,12-18:3 6,9,12-18:3--.+8,11,14..20:3 8,11,14.-20:3~5,8,11,14--20:4 5,8,11,14-20:4~ 7,10,13,16-22:4 (n - 3) Sequence 9,12,15-18:3--+6,9,12,15-18:4 6,9,12,15-18:4--,8,11,14,17-20:4 8,11,14,17-20:4---, 5,8,11,14,17-20:5 5,8,11,14,17-20:5--,7,10,13,16,19-22:5

0.21 2.54 0.40 1.14 0.27 2.46 0.34 1.31

that incorporate them into lipids, then the ratio of arachidonic acid to EPA should be a constant in all lipids. As shown in Table 5, this is not the case. It is clear from this Table that EPA is rather poorly incorporated into platelet PI and PS and liver PI. Majerus and his colleagues have shown that platelets contain two fatty acid activating enzymes. One enzyme is specific for acids, like arachidonic acid and EPA, which upon release from phospholipids are metabolized to eicosanoidsfl"33,47These investigators were able to show that 5,11,14-20:3 was an inhibitor of the arachidonic acid activating enzyme while the isomeric compound, 5,8,14-20:3, was a poor inhibitor?' Platelets metabolize 5,11,14-20:3 via an indomethacin sensitive pathway into 1l-hydroxy-5,12,14eicosatrienoic acid and 15-hydroxy-5,11,13-eicosatrienoic acid? This later observation directly supports the hypothesis that acids which are substrates for lipoxygenase and cyclooxygenase are activated by a specific activating enzyme. However, if activation specificity is the only regulatory step in dictating what acids are incorporated into phospholipids the ratios, as depicted in Table 5 for platelets, should be the same even though the absolute amounts of arachidonic acid and EPA may vary among individual phospholipids. This is not the case, suggesting that specificities also exist for incorporating acids into individual platelet phospholipids.

22

H. Spreeher

The results in Tables 1-5 show that, when equal amounts of the two esters were fed, linoleate was metabolized to arachidonate and acylated more readily than the analogous conversion of linolenate to EPA followed by its incorporation. In order to define whether these differences were due to specificities for fatty acid desaturation and chain elongation, we measured reaction rates with rat liver microsomes for analogous reactions in the (n - 3) and (n - 6) fatty acid biosynthetic pathways. These results, as depicted in Table 6, show that there were no major differences in reaction rates for analogous reactions in these two pathways. Rates of reactions, as measured with rat liver microsomes, thus cannot be used as a sole predictor to define what polyunsaturated fatty acids are produced for subsequent acylation into either liver or platelet phospholipids. Finally, it must be stressed that dietary fat change will modify the fatty acid composition of the same lipid from different cells in unique ways. For example (Table 4), there is no detectable 22:5(n - 3) plus 22:6(n - 3) in platelet PI when rats are maintained on a chow diet. When they are fed the linoleate or the linoleate-linolenate diet, these values were 3.6 and 1.7%, respectively. The amount of 22: 5(n - 3) plus 22:6(n - 6) in liver PI from chow fed rats was 2.9% and it increased to 21.2 and 9.1%, respectively, when rats were fed the linolenate or the linoleate-linolenate diet.

INCORPORATION OF ARACHIDONIC ACID AND (n-3) INTO HUMAN PLATELET PHOSPHOLIPIDS

FATTY

ACIDS

The results in Table 7 were obtained when five [l-14C]labeled fatty acids (10/ZM) were incubated for l hr with 9 x l0 s washed human platelets. 43These results compare the uptake of each substrate into individual phospholipids and, in addition, show that each acid is also chain-elongated with subsequent acylation of the chain-elongated product. Although platelets are not able to desaturate fatty acids, these results show that circulating fatty acids can be taken up by platelets and, in part, chain-elongated prior to acylation. PI had the highest specific activity when the substrates were 20-carbon acids. PI is characterized by its high level of arachidonic acid. 5'26Neither 18:3(n - 6) nor 18:4(n - 3) was a particularly good substrate for incorporation into PI. Once these two fatty acids were chain-dongated, the resulting 20-carbon acids were readily acylated into PI. The acids, 2 0 : 3 ( n - 9 ) , 20:4(n - 6) and 20:5(n - 3), all have their first double bond at position-5. The first double bond in 20:3(n - 6) and 20:4(n - 3) is at position-8. The results show that 20-carbon acids are incorporated into PI but recognition for specific 20-carbon acids is not absolutely dependent on the position of the first double bond. The results in Fig. 1 compare the incorporation of arachidonic acid and 20:5(n - 3) into individual platelet phospholipids. In these studies, 9 x l0 s platelets were incubated alone with either 10/zM [1-14C]arachidonic acid or [I-14C]20:5(n- 3) to compare their rates

TABLE 7. The Incorporation of Acids and Their Chain-Elongated Products into Human Platclets #tool fatty acid incorporated/tool o f phospholipid

Substrate

Fatty Acid

PI

PC

PS

PE

18:3(n--6)

18:3(n--6) 20:3(n - 6) 18:4(n -- 3) 20:4(n-3) 20:3(n--9) 22:3(n - 9) 20:4(n - 6) 22:4(n - 6) 20:5(n - 3) 22:5(n -- 3)

1672 1746 982 998 33305 791 34767 842 9931 918

1394 314 1309 151 4577 138 7811 128 3834 159

980 -431 62 1516 78 4111 58 1650 68

221 50 125 27 608 71 1884 310 973 189

18:4(n -- 3) 20:3(n--9) 20:4(n -- 6) 20:5(n - 3)

Washed plat¢lets (9 x 10S/ml) were incubated with I0/~M substrate for I hr. Platelet phospholipids were isolated, converted to methyl ester and separated by HPLC with acetonitrile at a flow rate of 0.5 ml/min. Results are averages of two separate experiments,

Metabolism of fatty acids

23

! ! 16

0

12

x m

!

PC

! 0 IO ,0

IO 15 I0 ,0 aO 0

0 I0

S iO

I0 15 iO ~'0 I0 0

0 5 IO I0

IO iS IO IO I0 0

0 IO

S I0 I$ IO IO I0

I0 ~ i aO 5 (n-3} 0 )/M204(fl-6)

FiG. 1. Simultaneousincorporationof [1-14C]arachldonicacid and [1-'(C]5,8,l 1,14,17-eicosapentaenoic acid into individualplatelet pho~holipi~. of acylation into individual phospholipids. Under these conditions, I0/~ M arachidonic acid was approximately substratc saturating relative to acylation. Experiments were then carried out in which 10/~M [l-~4C]arachidonic acid was simultaneously incubated with 5, 10 or 15/~M [1-1~']20:5(n -- 3). In all lipids, there was a concentration-dependent increase in the amount of 2 0 : 5 ( n - 3) incorporated which was accompanied by a decline in arachidonate acylation. The results in Figs 2 and 3 were carried out under identical conditions except that arachidonic acid was incubated with either [1-~-']22:5(n - 3 ) or [1-'~-']22:6(n- 3). Both of these 22-carbon (n - 3 ) acids were also incorporated into individual phospholipids, although not to the same extent as was 20:5(n-3). Again, at least with 22:5(n - 3), there was a decline in the amount of arachidonic acid incorporated as the concentration of this (n - 3) acid increased. Colelctivcly, these e x vivo studies suggest

60

PI

l:i ,2

o

PC

o

s

io is

io

o

s

IO

IO

I 0 IO

O

IO

IO IO IO

io

~

to

o

s

io

i~

io

•

s

0

I0

IO

I0

I0

0

IO

IO I0

io io IO

oo~dtlzzs(n-3) 0 JuM 1 0 4 I n ' S )

FK;. 2. Simultaneous incorporation of [l-'~]ar~hldo,4c lu~d and [l-'4C]7,10,13,16,19-docosa-

pentaenoic acid into individual piatelet phosphollpids.

24

H. Sprecher P! C2

i

| i

°iI PC

'_o x

! 0

I0

I0

I0

t5

I0

0

5

I0

15 I0

0

5

I0

15

I0

0

5

I0

15 I0 suM Z2~I In-3)

I0 I0

0

I0

I0 I0

I0 0

I0 I0

I0

I0

0

I0

I0

I0

I0

0 JiM Z 0 4 (n-6)

FIG. 3. Simultaneous incorporation of [1J4C]arachidonic acid and [l-~4C]4,7,10,13,16,19-docosahexaenoic acid into individual platelet phospholipids.

that, if the plasma concentration of long chain ( n - 3) acids is increased, they will effectively compete with arachidonic acid for acylation. It is not known whether these 22-carbon acids are activated by the same enzyme acting on arachidonic acid. When archidonic acid and 20: 5(n - 3) are activated the CoA derivative is, in most cases, the true substrate for acylation. In liver microsomes, the CoA derivative is both the substrate for malonyl CoA-dependent chain-elongation and the product of this reaction sequence. 2 If an analogous situation exists in platelets, then competition may exist between acylation and chain elongation followed by subsequent acylation. 22-Carbon acids could thus be incorporated into platelet phospholipids via a pathway that does not involve their direct activation. Arachidonic acid is one of the major polyunsaturated fatty acids in platelet PE. The above studies show that all acids are rather poorly incorporated into this lipid. Recent studies in Deykins laboratory describe the presence of two distinct transacylase activities in human platelets. 22.23 These pathways are of the most importance for synthesis of phosphatidylserine (PS) and the plasmalogenic form of (PE). (PC) is the substrate for both the CoA dependent and independent pathways. The low amount of fatty acid acylated into PE and PS may be due, in part, to lack of the required labeling of PC in order to observe significant incorporation of fatty acids into PE and PS.

METABOLISM OF LONG CHAIN (n-3) AND (n-6) POLYUNSATURATED F A T T Y A C I D S BY P L A T E L E T L I P O X Y G E N A S E A N D C Y C L O O X Y G E N A S E

When human platelets were incubated with either [1-14C]22:6(n - 3)' or [1-14C]22:5(n - 5 ) , 4 they were both metabolized via an indomethacin insensitive pathway into only two products. These metabolites were identified from their mass spectra as isomeric 14- and 1 l-hydroxy fatty acids. The ratio of the 14- to the I l-isomer produced from both acids was about 3:1. The results in Fig. 4 compare the substrate dependent metabolism of [1-14C]22:5(n - 3 ) into hydroxy fatty acids both in the absence and with 20/~M arachidonic acid. At low 2 2 : 5 ( n - 3 ) concentrations, there was an apparent stimulation in synthesis of both hydroxy fatty acids by arachidonic acid. This stimulatory effect was not blocked by indomethacin suggesting that 12-HETE or 12-HPETE may activate production of these hydroxy acids from 22:5(n - 3 ) . The results in Fig. 5 were obtained when increasing concentrations of [l-~4C]arachidonic acid were incubated with and without 20pM 2 2 : 5 ( n - 3). The inserts in this figure show that 2 2 : 5 ( n - 3), like 20: 5(n - 3) 3° and 22:6(n - 3)6.35inhibit the synthesis of thromboxane B2 and HHT. At low concentrations of arachidonic acid, in the presence of 20 t~t,I 22:5(n - 3), there was an apparent stimulation in the synthesis of 12-HETE. This is most likely due to shunting of

Metabolism of fatty acids

.

._

4

/

25

j ' ] ~

4

II-HDPI[

2

o

~

~

~

so o

~

so

~

[ t - ~4C] ~ : 5 (,~M)

FIG. 4. Platelets (l.5x108/0.5ml) were incubated with increasing concentrations of [i-14CJ7,10,13,16,19-docosapentaenoicacid without(O) and with (O) 20/~u arachidonicacid for 3 rain. Results are averagesand range of two separateexperiments. arachidonic acid into the lipoxygenase pathway because thromboxane B2 and HHT synthesis were partially blocked by 22:5(n - 3 ) . Collectively, these observations suggest that, if 2 2 : 5 ( n - 3 ) and arachidonic acid are released from platelet phospholipids, 2 2 : 5 ( n - 3) [and perhaps 22:6(n - 6 ) ] will inhibit thromboxane A2 and HHT synthesis resulting in increased production of 12-HETE. This compound or its hydroperoxy precursor may activate the synthesis of 14- and l l-hydroxy acids from 2 2 : 5 ( n - 3) and perhaps 22:6(n -3). The overall effect would be to increase the synthesis of hydroxy fatty acids in the platelet. We previously showed that 5,8,11,14-heneicosatetraynoic acid was a selective inhibitor of platelet lipoxygenase.4~ When various levels of this inhibitor were incubated with

16 2

I

12-

TXB2

IO

30

20[4(#M)

3o

CONTROL

/

12 - HETE

!i I

//

I 8"

2

HHT

4'

IO

0 w

i~

~

3o

20:4 (#i)

5O

so

D- ~c]-20:4 h.M) FIG. 5. Platelcts (l.5xlOS/O.5ml) were incubated with increasing concentrations of [l:4C]arachidonic acid without (O) and with (0) 20#M 7,10,13,16,19-docosapentaenoicacid.

26

H. Sprecher

i +] /~c]

?JO,~M

...../

/

""

J

]

ii" 0

•

*

I. SO

I0

-

?0

* I~'l

I0

ImUl"[S

FIG. 6. Reverse phase HPLC radiochromatogram when [1-~4C]7,10,I 3,16-docosatetraenoic acid was incubated with human platelets.

[l-~+C]arachidonic acid, the synthesis of 12-HETE was inhibited 50% by 0.5/~M acctylenic acid. Conversely, only 0.05/~M concentration of the acetylenic acid was required to inhibit the synthesis of both the 14- and 11-hydroxy acid isomers by 50%. These observations suggest that the lipoxygenase acting on 22:5(n - 3) and 22:6(n - 6) is different from the enzyme which metabolizes arachidonic acid to 12-HETE. If a single enzyme catalyzes synthesis of both the 14- and I 1-hydroxy acid isomers then, based on mechanistic studies done with arachidonic acid, ~7 protons must initially be abstracted from positions 12 and 9, i.e. the a~ll and 14 carbon atoms. Hamberg ~6 recently reported that 6,9,12-1g:3 was metabolized into 10-hydroxy-6,8-pentadecadienoic acid via an indomethacin sensitive pathway as well as to 10- and 13-hydroxy octadecatrienoic acid isomers via a lipoxygenase pathway. A purified reticulocyte enzyme has also been described which metabolizes arachidonic acid into isomeric 12- and 15-hydroxyeicosatetraenoic acids. 3'24 Figure 6 shows a reverse phase high pressure liquid chromatography (HPLC) radiochromatogram when [1-~4C]22:4(n- 6) was incubated with human platelets. The mass spectrum of compound I (Fig. 7) establishes its structure as dihomo-thromboxane B2 and shows that platelets like kidney microsomes 39 have a cyclooxygenase which metabolizes this (n - 6 ) fatty acid. Compound II was shown to be 14-hydroxy-7,10,12nonadecatrienoic acidfl When the metabolites eluting between 55--65 min, were fractionated by normal phase HPLC, several minor metabolites were detected. The major metabolite constituted about 90% of the total lipoxygenase products produced and was shown to be 14-hydroxy-7,10,12,16-docosatetraenoic acid. Although 2 2 : 4 ( n - 6 ) and 2 2 : 5 ( n - 3 ) are structurally similar, only 2 2 : 5 ( n - 3) is metabolized into two major lipoxygenase metabolites.

ioo. m4

i: I?'3 k.

. . )~e

111, _

dl~ m

m/I F I o . 7. M a s s s p e c t r u m

of compound

I in F i g . 6.

..

l

sm

m.~

Metabolism of fatty acids

27

C IO0O

D

13e,502 r,Wn 22:5( n..6

B

- I00

1(s4.3%) 80 I >-

5oo

I

sO

I

4o~

I

,< n*

20

I

0 I I0

0

I 20

I 30

I 40

I 50

I 6o

~

ELUTION TIME ( gIN )

FIo. 8. Reverse phase HPLC radiochromatogram when [l-~4C]4,7,10,13,16-docosapentaenoic acid was incubated with human platelets. TASL~ 8. Effect of 7,10,13,16-Docosatetraenoic Acid on the Metabolism of [l-14C]Arachidonic Acid by Human Platelets Rate (% of controls)

Substrate (~u) A. B. C. D. E.

[1-14C]20:4

22:4"

20 20 20 20 20

-4 10 20 20

TxBz 100(1.3) 8] (t.D 48 (0.6) 31 (0.4) 4(0.05)

HHT 100(1.7) 66(LD 52 (0.9) 25 (0.4) II (0.2)

12-HETE 100(6.1) 108 (6.6) 124 (7.6) 134 (8.2) 138(8.4)

Platelets (l.5xl0S/0.5ml) were incubated alone with 20#M [l-"C]arach/donic acid; nmols of products prodmxxl is shown in (). In B through D, plateletswere simultaneously incubated for 3 rain with the indicated concentrations of the two acids. In E, plateletswere incubated for 3min with 7,10,13,15-22:4 prior to the addition of [l-]'C]arachidonic acid. The incubation continued for an additional 3 min. Metabolites were separated by reverse phase HPLC.

Simultaneous incubation of 2 0 p M [1-1'C]arachidonic acid with increasing levels of 22:4(n - 6) resulted in a concentration-dependent inhibition in both thromboxane B2 and HHT synthesis (Table 8). In fact, the addition of 22:4(n - 6 ) actually resulted in an increase in 12-HETE synthesis. Incubation of platelets with 20~tM 22:4(n -6) for 3 rain prior to addition of arachidonic acid almost totally inhibited the synthesis of both cycloxygenase products with an apparent stimulation 12-HETE production. During the initial 3 rain incubation period, about 5 nmols of 14-hydroxy-7,10,12,16-docosatetraenoic acid were produced from 22:4(n - 6). It remains to be determined whether 12-HETE and the 14-hydroxy acid derived from 22:4(n - 6 ) are made by the same lipoxygenase. Figure 8 shows a reverse phase HPLC radiochromatogram when [1-'%~]22:5(n - 6) was incubated with platelets. Compounds A, B, and C were shown to be A4-dihomothromboxane B2, 14-hydroxy-4,7,10,12-nonadecatetraenoic acid and 14-hydroxy-4,7,10,12,16docosapentaenoic acid, respectively.2 The thromboxanes from both 22:4(n-6) and 22:5(n - 6 ) were produced and about 20% as rapidly as was thromboxane B2. SUMMARY

Arachidonic acid is the principal unsaturated acid in most membrane lipids. Membrane lipids also contain a variety of other (n - 6) and (n - 3) fatty acids. The amounts of (n - 6) and (n - 3 ) fatty acids in membrane lipids can be modified by dietary fat change. Our studies show that long chain ( n - 6) and ( n - 3) acids are metabolized by platelet lipoxygenase and cyclooxygenase. When cells are exposed to various agonists, a variety of unsaturated fatty acids may be released. Our studies show that they have the potential

28

H. Sprecher

of modifying physiological function both by mediating arachidonic acid metabolism and as direct precursors for oxygenated metabolites which themselves may interact with specific receptors to regulate biological processes. Acknowledgement--These studies were in part supported by Grants AM18844 and AM20387 from the National Institutes of Health.

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