the diet and, unfortunately, the drug was only able to reduce the cholesterol concentration by one-half as much compared to those who responded to both diet and drug therapy. Although HDL-cholesterolleveldecreased somewhat, the LDL fraction accounted for most of the decrease and this should reduce the risk to develop coronary artery disease. Although the investigators note that reducing cholesterol and saturated fat intake while increasing consumption of polyunsaturatedfat remains the first approach to treatment, some might argue about the rather high level of polyunsaturated fat in the diet given to all patients. Patientsprobably had to drink some of the corn oil prescribed in the diet. Other data indicated that saturated fat reduction is sufficient in these patients to bring about maximum reduction in blood cholesterol and high levels of polyunsaturated fat may only be

needed to increase the palatability of the diet. 0 1. J. Lelorier, S. DuBreuil-Quidoz, S. LussierCacan, Y-S. Huang and J. Davignon: Diet and Probucol in Lowering Cholesterol Concentrations. Additive Effects on Plasma Cholesterol Concentrations in Patients with Familial Type II Hyperlipoproteinemia. Arch. Int. Med. 137: 1429-1434, 1977 2. D.S. Fredrickson, R.I. Levy and M. Bonnell in The Dietary Management of Hyperlipoproteinemia: Hypercholesterolemia. P. 13. U.S.Department of Health, Education and Welfare and the National Heart and Lung Institute, Bethesda, 1974 3. E.R. Briones, P.J. Palumbo, B.A. Kottke, R.D. Ellefson and R.A. Nelson: Nutrition, Metabolism and Blood Lipids in Humans with Type Ila Hyperlipoproteinemia. Am. J . Clin. Nutrition 26: 259-263, 1973

THE MULTIPLE PATHWAYS OF ARACHIDONIC ACID METABOLISM The discovery of a new series of metabolites which are produced by the reaction of arachidonic acid with oxygen provides a new insight into factors controlling platelet aggregation, vasoconslriction and other biological processes.

Key Words: arachidonic acid, aspirin, platelets, prostaglandins, thromboxanes, vasoconstriction

Our knowledge of the multiple metabolic pathways that arachidonic acid may follow by reaction with molecular oxygen has advanced exceedingly rapidly in the last few years. The impetus to these advances stems from the earlier discovery of the role of the essential unsaturated fatty acids as precursors of prostaglandins. The prostaglandinsinitially isolated are relatively stable compounds. Prominent among the biological properties they display is their ability to regulate the production of cydic AMP and thus to possess hormone-like actions. For conveniencethese compounds will be referredto as the classical prostaglandinsin 10 NUTRITION REVIEWS I VOL. 36, NO. 1 JANUARY 1978

order to distinguish them from the newer and more unstable prostaglandins to be dealt with here. In addition to these new prostaglandins, arachidonic acid may give rise to nonprostaglandin compounds, thromboxanes and hydroperoxide derivatives. These recently discovered metabolites of arachidonic acid possess a variety of biological activities which are important in the control of platelet aggregation and vasoconstriction. The metabolic pathways producing these new metabolites of arachidonic acid, the ability of certain drugs induding aspirin to block some of thafe pathways, and the biological activity of some of the products formed are the subjects of this review. Arachidonic acid gives rise to the classical

prostaglandin, PGEz. Studies on the produc- blocked by the presence of arachidonic acid. tion of this prostaglandin led Hamberg, Sa- It was suggested by these workers that aspirin muelsson and their co-workersltz to the isola- acts as an active-site acetylating agent for the tion and identification of two precursor com- enzyme cyclo-oxygenase. The acetylation of cyclo-oxygenase by pounds, endoperoxides, which are lermed prostaglandin GZ (PGGz) and prostaglandin aspirin would not constitute an isolated case of HZ(PGHz). Formation of PGGzfrom arachido- acetylation by this drug. Aspirin has been prenic acid is catalyzed by an enzyme called fatty viously shown by Pinckard et al.9 to acetylate acid cydo-oxygenase or prostaglandin syn- nucleic acids and a large variety of proteins thaw. PGH2 is apparently formed from PGGz including enzymes and hormones. In these by a nonenzymatic reaction. In turn it may be studies aspirin, radioactively labeled in the converted to PGEz by the action of an isomer- acetyl group, was incubated in vitro with the ase. These two new compounds were found to various compounds employed for 24 hours be much more potent than PGEz in producing at 37°C. The concentration of aspirin was aggregation of human platelets or contrac- 500pM. The extent to which different proteins tion of the rabbit aorta. This property of the are acetylated would appear tovary widely. The endoperoxides, as will be discussed later, is effect of acetylation on the biological properdue to their ability to act as precursors of com- ties of the substances was not undertaken in pounds other than PGEz.The Swedishworkers these studies. In the case of cyclo-oxygenase also showed that aspirin and indomethacin the inhibitory action of aspirin would appear to inhibited the production of the endoperoxides. occur within minute$?* at 37% in vitro and These results suggested that the previously at concentrations of aspirin as low as 2pM. observed ability of aspirin to block prosta- It may be noted that in humans the plasma glandin synthesis3v4was due to its inhibition level of aspirin reaches values well above 500pM and remains there for hours after the of the enzyme cyclo-oxygenase. Evidence is now available that indicates ingestion of a single 2 g dose of aspirin.l0 aspirin can acetylate fatty acid cyclo-oxygen- Human platelet production of prostaglandin as8 and irreversibly inhibit it. Studies516 prior has been shown to be markedly affected one to the discovery of the endoperoxides showed hour after the ingestion of 600 mg (twotablets) .~ it seems reasonable to that the inhibition of platelet function by aspirin of a ~ p i r i n Thus was accompanied by incorporationof radioac- condude that the ingestion of moderate doses tivity into platelets when aspirin labeled with of aspirin by humans may result in the acetylaa radioactive acetyl group was employed. tion of proteins and other compounds. In the Roth and c o - ~ o r k e r sstudied ~ ~ ~ the action of case of cydo-oxygenase its acetylation may radioactive aspirin on human platelets and account for not only some of the therapeutic bovine and ovine seminal vesicles. An in- but also unwanted side effects of aspirin. It soluble protein fraction from these tissues remains to be determined whether cyclocontaining radioactivity was solubilized with oxygenase is unusually sensitive to acetylasodium dodecyl sulfate and submitted to poly- tion by aspirin and the nature of the specific acrylamide gel electrophoresis. Radioactivity active site that is acetylated. The recent was found to reside in a single peak which was results of Roth et a1.l' would suggest that not estimated to be a protein with a molecular more than one site on the enzyme is attacked. weight of 85,000. All three tissues yielded a These workers purified the protein which besimilar radioactive product. The extent of in- comes labeled when radioactive aspirin corporationof radioactivityinto this proteinwas inactivates the cyclo-oxygenase of sheep shown to parallelthe inhibitionof endoperoxide vesicular gland. This protein is reported to formation as measured by the formation of contain 0.5 mole of acetyl residues per mole malondialdehyde, a breakdown product of the *of protein. The finding that the endoperoxides PGGz endoperoxides. Incorporation of radioactivity occurred only when the acetyl portion of as- and PGHzwere much more potent agents than pirin was labeled and incorporation could be PGEz in stimulating platelet aggregation and NUTRITION REVIEWS I VOL. 36, NO. 1, I JANUARY 1978 11

vasoconstriction suggested a biological role per se for these compounds in such processes. Further investigations of this activity of the endoperoxides led to the discovery that they were not only the precursors of the classical prostaglandins but could also follow a second metabolic pathway.12This pathway resulted in the formation of nonprostaglandin derivatives which are called thromboxanes. An enzyme named thromboxane synthetase converts PGH2 into thromboxane A2 (TXA2). In aqueous solutions TXA2 is reported-to have a half-life of 32 seconds at 3PC, reacting with water to form a more stable compound thromboxane 8 2 (TXB2) which is apparently without biological activity. On the other hand TXA2 has the ability to induce platelet aggregation and cause vasoconstriction, properties displayed by its precursor PGH2. The question has thus arisen as to whether the activity exhibited by PGH2 is entirely due to its conversion to TXA2. Evidence that such a conversion must take place before aggregation of human platelets occurs has now been presented. Gorman and co-workers13 synthesized an azeprosta-glandin analog, 9, 11 azo-prosta-5,1S dienoic acid. They report that platelet aggregation induced by arachidonate or PGH2 is prevented by this compound. Concomitantly production of the stable thromboxane 6 2 is prevented and an enhanced production of prostaglandin E2 occurs. These actions of the azo analog thus indicate that it is a specific inhibitor of thromboxane synthetase. In addition to the formation of prostaglandin E2 or thromboxanes, the endoperoxides produced from arachidonic acid may follow a third pathway. This results in the formation of a compound first called prostaglandin X and subsequently referred to as prostacyclin or prostaglandin 12 (PG12).14*15 It is formed .by the action of an enzyme which is present in the microsomesof arterial walls but not in platelets. It is unstable and converted into an apparently inactive but more stable compound, 6 ketoPGFla. Prostaglandin 12 causes vasodilatation and is a potent inhibitor of platelet aggregation. Thus it has an action which is the exact opposite of that displayed by thromboxane A2. The possible interplay of TXA2 and PG12 may be visualized in the following manner. Plat12 NUTRITION REVIEWS I VOL. 36, NO. 1 JANUARY 1978

elets which are forming the endoperoxides and producing TXA2 will attempt to stick to blood vessel walls and form thrombi. However, endoperoxides which escape from the platelets or are formed in the endothelium of vessel w a l W may be converted to PG12 which will inhibit thrombi formation. At the same time the vasoconstrictive action of TXA2 will be counteracted. It may be speculated that events such as plaque formation or erosion of vessel walls will prevent or retard the formation of PGh and thus favor thrombi formation and hypertension. A specific inhibitor of PG12 formation, tranylcypromine, has been described.lS Thus synthetic compounds are available which will specifically block the production of PG12 or T a 2 . The possible therapeutic use of these compounds as drugs to channel endoperoxides into one or the other of these substances which possess opposing physiological properties remains to be tested. It is obvious, however, that the ability to control these two different metabolic pathways of arachidonic acid could be important in the management of various disease states in humans which involve vasoconstriction and platelet aggregation. The final pathway of arachidonic acid metabolism that needs consideration is its transformation to hydroperoxide derivatives. Hamberg and Samuelsson2 showed that incubation of human platelets with arachidonic acid led not only to the production of the endoperoxides but also to the formation of 12Lhydroperoxy-5,8,10,14 eicosatetraenoic acid (HPTE). This compound is converted by platelets or by SnCh reduction in vitro to 12Lhydroxy-5,8,10,14 eicosatetraenoic acid (HETE). The enzyme in platelets which catalyzes the production of HPETE is a lipooxygenase which is novel in that unlike other lipooxygenases its attack is upon the (2-12 group of arachidonic acid. Recently Hammarstrom and Fdardeau17 carried out the separation of the prostaglandin endoperoxide synthase and thromboxane synthase of human platelets. They report that HPETE b m o t HETE inhibited the latter enzyme and suggest that HPETE might serve as a regulator of thromboxane synthesis in vivo.

Another role for unsaturatedfatty acid perox- ity of arachidonic acid metabolites to influence the adenylate cyclase system on one hand ides is suggested by the work of Hidaka and and the guanylate system on the other. It is Asano.1sThese workers report that the stimuladifficult to think of another dietary substance tion of the guanylate cydase system of human that follows so many diverse metabolic pathplatelets that is observed upon the addition of unsaturated fatty acids is due to their conver- ways as does arachidonic acid and which at the same time produces metabolites with such sion to peroxides. They incubated emulsions of a wide range of biological activities. No matter arachidonic acid with soy bean lipoxidase to what the future holds, arachidonic acid is form peroxides. When peroxides so formed indeed to be classed in more ways than one as were added in increasing amounts to a prepessential. 0 aration of the platelet guanylate cyclase system they observed a corresponding in1. M. Hamberg, J. Svensson, T. Wakabayashi crease in the activity of this system. This acand B. Sarnuelsson, Proc. Nat. Acad. Sci. USA tion of fatty acid peroxides, which are presum71:345349,1974 ably not identical with HPETE, is attributed to 2. M. Hamberg and B. Sarnuelsson, Proc. Nat. their oxidationof sulfhydrylgroups of guanylate Acad. Sci. USA 71:3400-3404,1974 cyclase. 3. J.R. Vane, Nature (New Biol.) 231: 232-235, 1971 Thus there are two separate pathways that 4. J.B. Smith and A.L. Willis, Nature (New Biol.) arachidonic acid may follow by reaction with 231:235237,1971 oxygen. Whether biological factors exist that 5. H. Al-Mondhiry, A.J. Marcus and T.H. Spaet, determine the amount of arachidonic acid Proc. SOC.Exp. Med. 133:632-636,1970 entering one or the other of these pathways 6. F.J. Rosenberg, P.E. Gimber-Phillips, G.E. remains to be determined. It is possible, howGroblewski, C. Davison, D.K. Phillips, S.J. ever, by the use of aspirin or indomethacin Goralnick and E.D. Cahill, J. Pharm. Exp. to block the pathway leading to the endoperoxTherap. 179:410-418,1971 ides without interfering with the production 7. G.J. Roth and P.W. Majerus, J. CIin. Invest. of the hydroperoxides.Both pathwaysgive rise 56:624-632,1975 to metabolites that are unstable but which 8. G.J. Roth, N. Stanford and P.W. Majerus, Proc. Nat. Acad. Sci. USA 72:3073-3076,1975 possess biological activity. This instability of 9. P.N. Pinckard, D. Hawkins and R.S. Farr, these metabolites imposes a built-in limitation Nature 219:68-69,1968 on the duration of their action. Over and above 10. P.K. Smith, H.L. Gleason, C.G. Stoll and S. this type of inherent control there is the remarkOgorzalek, J. Pharm. Exp. Therap. 87: 237able fact that one metabolite of arachidonic 255,1946 acid may possess the ability to either block 11. G.J. Roth, N. Stanford, J.W. Jacobs and P.W. the production or counteract the biological Majerus, Biochemistry 16:4244-4248,1977 activity of another. This sort of interplay is to 12. M. Hamberg, J. Svensson and B. Samuelsson, be seen in the platelets where a metabolite of Proc. Nat. Acad. Sci. USA 72:29942998,1975 the hydroperoxide pathway, HPETE, is re13. R.R. Gorman, G.L. Bundy, D.C. Peterson, F.F. portedto control the productionof a metabolite, Sun, O.V. Miller and F.A. Fitzpatrick, Proc. Nat. Acad. Sci. USA 74:4007-4011,1977 TXA2, formed in the other pathway. The most 14. S.Moncada, R. Gryglewski, S. Bunting and J.R. striking example of this interplay is perhaps Vane, Nature 263:663665,1976 provided by the two metabolites thromboxane 15. R. Gryglewski, S. Bunting, S. Moncada, R.J. A2 and prostaglandin 12 both of which are Flower and J.R. Vane, Prostaglandins 12: formed as a result of the entrance of arachide 685713,1976 nic acid into the endoperoxidase pathway. 16. B.B. Weksler, A.J. Marcus and E.A. Jaffe, Here, however, thromboxane A2 with its ability Proc. Nat. Acad. Sci. USA 74: 3922-3926, to aggregateplatelets and cause vasoconstric1977 tion is formed in one tissue, the platelets, w. 17. s. Hamrnarstrom and P. Falardeau, Proc. Nat. while prostaglandin 12 with its opposing action Acad. Sci. USA 74: 3691-3695,1977 is formed in another tissue, the arterial walls. 18. H. Hidaka and T. Asano, P~OC.Nat. Acad. sci. USA 74: 3657-3661,1977 In addition one may consider the reported abilNUTRITION REVIEWS I VOL. 36, NO. 1 , I JANUARY 1978 13