EXPERIMENTAL NUTRITION

DIETARY ESSENTIAL FATTY ACIDS, PROSTAGLANDIN FORMATION AND PLATELET AGGREGATION Feeding ethyl arachidonate to human volunteers increased the arachidonate levels of plasma and platelet phospholipids and the sensitivity of the platelets to aggregation. The prostaglandin endoperoxides, which are precursors of prostaglandins, can induce platelet aggregation. These are formed when platelets are aggregated in vitro by thrombin.

and fat. The linoleate content was 8 to 10 percent of dietary fatty acids. The proportion of dietary fat was not stated, but if fat supplied Thomasson' suggested that prostaglandins 40 percent of the dietary energy as is usual (PG) may play an important role in the effect in American diets, then linoleate supplied 3 to of dietary fat on the development of athero4 percent of the energy. Arachidonate at a level sclerosis and thrombosis since PG are formed of 6 g (54 kcal) would have supplied 2 percent from the dietary essential fatty acids. The efof the dietary energy at an intake of 2500 kcal fect of PG in the etiology of atherosclerosis (ca 10,000 kJ). All subjects showed a decrease may be exerted through processes regulated in the threshold concentration of ADP needed by PG, including platelet adhesion and agto cause irreversible aggregation of plateletgregation, vasoconstriction and vasodilatation. rich plasma. In view of this increased aggregaThomasson also proposed that consumption tion, the arachidonate supplementation was of excess linoleic acid should increase in vivo halted. The threshold dose of ADP for platelet synthesis of PG. aggregation was again normal two weeks after Hwang and co-workers2 demonstrated that discontinuing the arachidonate supplement. the serum concentration of PGEl and PGFz,, The urinary excretion of PG metabolites inwere higher in rats fed a linoleate-rich diet than creased during the supplementation period, in rats fed a diet supplying the same level of evidence that PG formation increased in this fat, but a lower intake of linoleate. period. Arachidonate supplementation doubled Adhesion of blood platelets to collagen in the basement membrane of the blood vessel the levels of arachidonate in plasma phosphostarts a process of secretion from the sublipids, in comparison with values for the concellular granules of the platelets, i.e, the release trol period before supplementation (32 percent reaction, which leads to platelet aggregati~n.~ cf 16 percent). Linoleate showed correspondCollagen may be the most platelet-reactive ing decreases. The proportion of arachidonate substance in the vessel wall. This aggregation declined after discontinuing the supplement. probably accounts for the primary arrest of Sixteen days later, arachidonate levels in bleeding after disruption of the vessel wall. The plasma phospholipids, triglyceride and free primary aggregation can be started by ADP, fatty acids returned to presupplementation thrombin, epinephrine or serotonin. values, although the level in cholesterol ester The potential importance of the level of platewas still higher. let arachidonate on platelet aggregation has In fatty acids of platelet phospholipids, the been shown recently by Seyberth and coproportion of arachidonate increased, but the w o r k e r ~ .They ~ fed ethyl arachidonate (6 g increases were not so great as in plasma per day) to male volunteers for two to three phospholipids. The initial level of arachidonate before supplementation was more than 20 weeks. The diets were kept isocaloric and had a constant proportion of carbohydrate, protein percent, and supplementation increased the Key Words: prostaglandins, arachidonate, aggregation, fatty acid, platelet, phospholipid

NUTRITION REVIEWS I VOL. 34, NO. 8 I AUGUST 1976 243

values by 5 to 6 percent. The greatest increase occurred in cholesterol ester arachidonate, which showed a two- to threefold increase (4 percent to 12 percent or 8 percent to 16 percent). Linoleate decreased in platelet lipids as arachidonate increased. At four to ten days after discontinuing the supplement, platelet phospholipid arachidonate decreased, and linoleate increased. The arachidonate levels were still higher, however, than in the presupplemental period. The prostaglandin endoperoxides, prostaglandin G2 (PGGz) and prostaglandin H Z (PGHz) are intermediates in the synthesis of PG from arachidonic acid.5 Nonesterified arachidonate (“free” arachidonate) is the precursor of the endoperoxide intermediates, which can induce platelet aggregation. The release of arachidonic acid from phospholipids is an initial stage in the synthesis of the endoperoxides during platelet aggregati0n.~9’Platelets normally contain little free arachidonic acid although the levels of arachidonate in platelet phospholipids are quite high.* The utilization of platelet phospholipid arachidonate for PG formation has been investigated by Bills and c o - w ~ r k e r sThey . ~ first incubated human platelets with 14C-arachidonic acid to label the phospholipids. These labeled platelets were then used for studies of formation of 14C-labeled PG intermediates, in response to platelet aggregation induced by thrombin. When platelets were incubated with 14Carachidonic acid, the incorporation of labeled fatty acid increased with incubation time, with 90 percent of the label entering the phospholipids. A small amount of 14C was incorporated into triglycerides. After incubation, the platelets were washed to remove nonesterified 14C-arachidonate,and any other nonesterified radioactive products which might have been formed. Very little PG formation or very little oxidation of arachidonate to short-chain fatty acids occurred during these procedures. Over 95 percent of the unesterified 14C in the platelet washings was arachidonic acid. Different preparations of platelets varied in the capacity to incorporate 14C-arachidonic acid. Nevertheless, the relative uptake by the different lipid classes was reproducible with 244 NUTRITION REVIEWS I VOL. 34, NO. 8 / AUGUST 1976

different lots of platelets. The neutral lipid fraction contained 1 to 4 percent of the radioactivity, chiefly as nonesterified fatty acid. Most of the radioactivity was present in the phospholipid fraction, with 56 percent of the radioactivity in phosphatidylcholine (PC), 10 percent in phosphatidylethanolamine (PE), 14 percent in phosphatidylinositol (PI) and 7 percent in phosphatidylserine (PS). This pattern of 14Carachidonate incorporation into the phospholipid classes differed from the distribution of arachidonate, as a percent of the fatty acids in the various phospholipid classes. The highest proportion of arachidonate (percent of fatty acids) occurred in PE, which showed the lowest incorporation of 14C-arachidonate.When Bills and co-workers calculated the moles of arachidonate per mole of phospholipid for each of the phospholipid classes, PE had the highest value, followed by PI, PS and PC. When the moles of incorporated 14C-arachidonate per mole of phospholipid were calculated, however, PI had the highest value, followed by PC, PS and PE. The ratio of 14C-arachidonatein PC to 14C-arachidonatein PI was similar to the ratio of arachidonate in PC (nmoles per pmole PC) to arachidonate in PI (nmoles per pmole PI). The similarity of these two ratios is evidence that incorporation of 14C-arachidonate by the incubated platelets followed the pattern of arachidonate incorporation which occurs in vivo. When similar calculations were made to compare the PC values to the corresponding PE and PS values, however, the 14C-incorporation ratios were much lower than the ratios based on nmoles arachidonate per pmole PE or PS. Suspensions of labeled platelets were then incubated with thrombin to cause platelet aggregation. Incubation at 37°C for five minutes caused a 20 to 40 percent decrease in PC radioactivity and a 50 percent decrease in PI radioactivity. PS showed a 10 percent decrease, but no significant changes occurred in PE radioactivity. The decreased radioactivity in phospholipids was accompanied by formation of 14C-labeled, oxygenated products of arachidonic acid. These compounds were not formed in the absence of thrombin and were identified as products of both PG cyclo-oxygenase and lipo-oxygenase activity. The

amount of 14C as nonesterified arachidonic acid also increased somewhat. When the labeled platelets were treated with indomethacin before adding thrombin, there was a decrease in two of the PG metabolites, but a third metabolite increased. The amount or the pattern of 14C-arachidonaterelease from phospholipids was not altered by indomethacin. lndomethacin has been shown to inhibit cyclo-oxygenase activity without changing the release of arachidonic acid.l0>11 When the platelets were incubated with eicosatetraenoic acid, thrombin addition produced chiefly an accumulation of 14C-arachidonic acid, with much less of the other products. Eicosatetraenoic acid inhibits both the cyclooxygenase reaction, the initial step in formation of PG endoperoxides and PG, as well as the lipoxygenase reaction that forms hydroxyeicosatetraenoic acids, which are not PG precursors. The total amount of radioactivity released from the platelets treated with eicosatetraenoic acid plus thrombin was also less than with the thrombin-treated platelets. The release of arachidonic acid, however, was still chiefly from PC and PI. When the platelets were suspended in medium containing 3 percent serum albumin and thrombin was then added, there was a 50 percent reduction in the amount of 14C released from PC and PI. The 14C-oxygenated products of arachidonic acid also decreased, in comparison with products formed when thrombin was added without albumin. There was a large increase, however, in nonesterified arachidonic acid. The accumulation of free arachidonic acid by platelets incubated with thrombin in the presence of serum albumin may be similar to events in vivo. Albumin presumably remained extracellular and did not enter the platelets. Thus, an accumulation of arachidonate in the medium indicates that a considerable amount of the arachidonate released from the phospholipids was also released from the platelets. If such release occurs in vivo, this arachidonate may then be re-esterified into phospholipids. The increases in platelet arachidonate after feeding arachidonate to human subjects were relatively small. Yet the sensitivity of these plat-

elets to aggregation was definitely higher. It remains to be determined whether (a) the increased platelet aggregation was the consequence of the increase in arachidonate, the decrease in linoleate or other factors, and (b) whether high linoleate intakes, which will raise tissue linoleate as well as arachidonate, would also increase the sensitivity of the platelets to aggregating stimuli. Silver and co-workersl* showed that di-homogamma-linolenic acid, the precursors of the monoenoic PG series (PGEl), does not produce platelet aggregation in vitro. 0 1. H.J. Thomasson: Prostaglandins and Cardiovascular Diseases. Nutrition Reviews 27: 67-69, 1969 2. D.H. Hwang, M.M. Mathias, J. Dupont and D.L. Meyer: Linoleate Enrichment of Diet and Prostaglandin Metabolism in Rats. J . Nutrition 105: 995-1002, 1975 3. H.J. Weiss: Platelet Physiology and Abnormalities of Platelet Function. New Engl. J . Med. 293: 531-541, 1975 4. H.W. Seyberth, O.Oelz, T. Kennedy, B.J. Sweetman, A. Danon, J.C. Frolich, M. Heimberg and J.A. Oates: Increased Arachidonate in Lipids after Administration to Man: Effects on Prostaglandin Biosynthesis. Clin. Pharm. Therap. 18: 521-529, 1975 5. M. Hamberg, J. Svensson, T. Wakabayashi and B. Samuelsson: Isolation and Structure of Two Prostaglandin Endoperoxides that Cause Platelet Aggregation. Proc. Nat. Acad. Sci. USA 71 345-349, 1974 6. W.E.M. Lands and B. Samuelsson: Phospholipid Precursors of Prostaglandins. Biochim. Biophys. Acta 164: 426-429, 1968 7. H. Vonkeman and D.A. van Dorp: The Action of Prostaglandin Synthetase on 2-ArachidonylLecithin. Biochim. Biophys. Acta 164: 430-432, 1968 8. A.J. Marcus, H.L. Ullman and L.B. Safier: Lipid Composition of Subcellular Particles of Human Blood Platelets. J . Lipid Res. 10:108-114, 1969 9. T.K. Bills, J.B. Smith and M.J. Silver: Metabolism of [14C] Arachidonic Acid by Human Platelets. Biochim. Biophys. Acta 424: 303314, 1976 10. M. Hamberg and B. Samuelsson: Platelet Endoperoxides. Novel Transformations of Arachidonic Acid in Human Platelets. Proc. Nat. Acad. Sci. USA 71 : 3400-3404, 1974 NUTRITION REVIEWS / VOL. 34, NO. 8 / AUGUST 1976 245

11 C. Malmsten, M. Hamberg, J. Svensson and B. Samuelsson: Physiological Role of an Endoperoxide in Human Platelets: Hemostatic Defect Due to Platelet Cyclo-Oxygenase Deficiency. Proc. Nat. Acad. Sci. USA 72: 1446-1450, 1975

12. M.J. Silver, J.B. Smith, C. lngerman and J.J. Kocsis: Arachidonic Acid-Induced Human Platelet Aggregation and Prostaglandin Formation. Prostaglandins 4: 863-875, 1973

INFLUENCE OF PROTEIN RESTRICTION ON IMMUNE RESPONSE TO ALLOANTIGENS AND HOMOGRAFT REJECTION Experimental data on the relationship between protein intake and immune response to cells and tissues of different histocompatibility antigens are reviewed. Key Words: protein deficiency, cellular immunity, histocompatibility antigens, transplantation

Diets inadequate in calories, protein or other essential constituents often exacerbate infectious disease.’ One of the pathogenetic mechanisms for this is the marked alteration in host immunity associated with nutritional deprivation in man and in animals.* Protein deficiency reduces the incidence of spontaneous tumors3 and increases resistance to transplantable tumor^.^ This observation has been explained by the suggestion that ingestion of diets containing moderate amounts of protein is associated with a preferential suppression of tumor-specific “blocking” antibody, thereby resulting in heightened cell-mediated immune responses against tumor antigens.5 Malave and Layrisse6 investigated the differential effect of protein restriction on immunoglobulin M and G antibody response to histocompatibility (H-2) antigens upon primary and secondary stimulation with allogenic cells in mice. Inbred mice of C57BL/6 strain were weaned at 21 days after birth and fed ad libitum with a protein deficient diet containing casein in a concentration of 8 g percent. After six to seven weeks, the animals were injected intraperitoneally with lo7 spleen cells of DBA/2 strain which differed on the H-2 locus from the recipient’s cells. Employing ascitic tumor line L5178Y as target cells, the number of spleen cells forming alloantibodies was estimated in a plaque forming cell assay system, in which individual antibody forming cells appear as distinct areas of lysis or clear zones when viewed against strong direct light. In the 246 NUTRITION REVIEWS / VOL. 34, NO. 8 I AUGUST 1976

protein-deprived group, there was a reduction in the total number of spleen cells and in IgG antibody forming lymphocytes, whereas the proportion of IgM alloantibody plaque forming cells was increased. The serum hemagglutinin titers were similar or higher during the primary response and decreased during the secondary response. Thus there was a preferential suppression of cell populations involved in the IgG response to alloantigens. An impaired feedback inhibition or a reduction in suppressor cells may explain the unchanged or higher IgM response. Since pair feeding was not attempted and the animals fed protein-deficient diets consumed a lower amount of food than mice receiving a normal diet, the specific effect of protein and calorie deficiency is uncertain. A related aspect of cellular immunity is the ability to reject transplanted tissue from a genetically unrelated donor. Purkayastha and coworkers7 evaluated the influence of protein deficiency on homograft rejection. Six-weekold albino rats were fed a diet containing 3 percent casein for three to four weeks. The rate of rejection of skin homografts from another strain incompatible at a major locus was used as an index of cell-mediated immunity. Protein restriction did not alter the mean survival time of homografts, in contrast to earlier studies which showed a more rapid rejection of skin grafts in deprived mice.8 One limitation of this study was the relatively short duration of nutritional deprivation. It is known that the long lived small lymphocytes, the key cells in thymusdependent cell-mediated immunity, are reduced only after prolonged protein deficiency, 70 days in one study.9 Moreover, functionally dif-

Dietary essential fatty acids, prostaglandin formation and platelet aggregation.

EXPERIMENTAL NUTRITION DIETARY ESSENTIAL FATTY ACIDS, PROSTAGLANDIN FORMATION AND PLATELET AGGREGATION Feeding ethyl arachidonate to human volunteers...
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