Role of cytochrome P4502E1 in alcoholic liver disease pathogenesis

The intragastric tube feeding model is ideal for the study of the role of dietary factors and the effect of drugs on experimental alcoholic liver disease (ALD), since the model allows us to study the effect of a single variable in the diet on the pathology of liver where the blood alcohol level (BAL) is maintained over 150 mg%. By varying the dietary fatty acid composition we showed that the pathology was worsened by increasing linoleic acid or polyunsaturated fatty acids (PUFAs) in the diet where cytochrome P4502E1 (CYP2E1) was increased posttranslationally by high BAL. Concomitant with the increase in CYP2E1 there was evidence for an increase in lipid peroxidation (LP) by microsomes. Protein adducts of the products of LP were increased in the blood. Isoniazid (INH) enhanced this process and the pathology of ALD when INH was fed at therapeutic levels with ethanol. Preliminary studies show that diallyl sulfide, which inhibits and destroys liver CYP2E1 selectively, also modified the pathologic effects of ethanol. Thus we postulate that CYP2E1 induction plays a central role in the pathogenesis of ALD.     

SYNTHESIS OF POLYUNSATURATED FATTY ACIDS IN VITRO

Dehydrogenation and chain elongation of linoleic acid (18:2w6) by rat liver microsomes in vitro was inhibited by linolenic acid (18:3w3) and oleic acid (18:1w9). These results support the hypothesis, based on nutritional experiments, of a competition among linoleic, linolenic, and oleic acids in the reactions leading to the synthesis of more highly unsaturated fatty acids.     

Cyclic fatty acid monomers from heated oil modify the activities of lipid synthesizing and oxidizing enzymes in rat liver

Cyclic fatty acid monomers purified from a heated linseed oil were given for 2 wk to adult rats as triacylglycerol at two dose levels, i.e., 0.1 and 1 g/100 g diet, to determine their effect on some aspects of lipid metabolism. Indirect evidence of a peroxisome proliferator-like effect was observed, as determined by an elevation of some characteristic enzyme activities, such as peroxisomal acyl-CoA oxidase, and the microsomal omega- but also (omega-1)-laurate hydroxylase (CYP4A1 and CYP2E1, respectively). The dietary cyclic fatty acids induced a coordinated regulation between the activities of the lipogenic enzymes studied (Delta9-desaturase, phosphatidate phosphohydrolase) and peroxisomal oxidation, but not with mitochondrial beta-oxidation. The dose-dependent decrease of Delta9-desaturase activity (P < 0.05) with cyclic fatty acid monomer intake was accompanied by a similar decrease of the monounsaturated fatty acid level in liver. The increase in the gamma-linolenic acid level also suggested an increase in Delta6-desaturase activity with cyclic fatty acid intake (P < 0.05). In addition, our results strongly suggested that the altered liver levels of eicosapentaenoic and arachidonic acids were due to the peroxisomal retroconversion process in rats fed cyclic acids. Finally, an effect of these cyclic compounds on the carbohydrate metabolism cannot be disregarded because they decreased liver glycogen concentration. We conclude that cyclic fatty acid monomers affect different aspects of lipid metabolism, including a phenotypic peroxisome proliferator response. This provides the ground for further studies investigating the biochemical pathways that underlie the nutritional effect of such molecules.     

Dietary fish protein lowers blood pressure and alters tissue polyunsaturated fatty acid composition in spontaneously hypertensive rats

OBJECTIVE: To investigate the effect of two types of dietary protein on blood pressure, liver fatty acid desaturation and composition, and urine 6-keto-prostaglandin-F (PGF(1alpha)) level, the metabolite of prostacyclin. METHODS: 5-wk-old spontaneously hypertensive rats were fed 20% casein or purified fish protein. The fat source was 5% ISIO oil, which contains 47.9% (omega-6) and 1.7% (omega-3) total polyunsaturated fatty acids. After 2 mo on the diet, systolic blood pressure was reduced with fish protein compared with casein (189.8 +/- 10.5 versus 220.7 +/- 8.7). RESULTS: Excretion of 6-keto-PGF(1alpha) in urine was negatively correlated with blood pressure. Liver cholesterol and phospholipid concentrations were 1.71- and 1.27-fold lower with fish protein than with casein, respectively. The fish protein diet lowered the 20:4(omega-6) proportion and the ratio of 20:4(omega-6) to 18:2(omega-6) in liver microsomal lipids and phospholipids, which was due to the reduced microsomal Delta6(omega-6) desaturation activity. Dietary protein source did not affect omega-3 fatty acid composition, and this was associated with a similar activation of Delta6(omega-3) desaturation in liver microsomes. CONCLUSIONS: The present data indicated a significant blood pressure-lowering effect caused by fish protein, rather than by casein, that modified the fatty acid composition of liver phospholipids and liver microsomal total lipids.     

Postprandial serum triacylglycerols and oxidative stress in mice after consumption of fish oil, soy oil or olive oil: possible role for paraoxonase-1 triacylglycerol lipase-like activity

OBJECTIVE: Postprandial triacylglycerols and oxidative stress responses are influenced by the type of fat consumed. We investigated the effect of individual unsaturated fatty acids or oils (fish, soy, or olive) on postprandial triglyceridemia response in association with serum resistance to oxidation and paraoxonase-1 (PON1) activity. METHODS: Balb/C mice were supplemented with phosphate buffered saline (control), docosahexaenoic acid (omega-3), linoleic acid (omega-6), or oleic acid (omega-9; 500 mug/300 muL of phosphate buffered saline) and with fish, soy, or olive oil (300 muL); blood samples were collected 2 h after feeding. RESULTS: Serum triacylglycerol and oxidative stress responses increased after intake of all unsaturated fatty acids and oil supplements. However, ingestion of fish oil or its major fatty acid, docosahexaenoic acid, induced the most remarkable increase in postprandial serum triacylglycerols and in the susceptibility of serum to in vitro oxidation. Serum PON1 activity was decreased by 24% after fish oil ingestion. The increase in postprandial serum susceptibility to oxidation was lower after soy oil supplementation to PON1-transgenic mice in comparison with Balb/C mice, showing that PON1 attenuates the postprandial serum oxidative response. In parallel, in PON1-transgenic mice, a decreased postprandial triacylglycerol response was noted, suggesting PON1 involvement in triacylglycerol metabolism. PON1 exhibited a triacylglycerol lipase-like activity on chylomicrons. CONCLUSION: PON1 attenuates the postprandial oxidative stress response, and this could have resulted from PON1 lipase-like activity on chylomicron triacylglycerols.     

Effect of ursodeoxycholic acid on prostaglandin metabolism and microsomal membranes in alcoholic fatty liver.

The aim of the present study was to evaluate the effect of ursodeoxycholic acid (UDCA) on prostaglandin and fatty acid metabolism and the possible relation of these substances to the development of alcoholic fatty liver in rats. The effects of UDCA (40 mg/kg/day, 30 days) were studied in rats pair-fed a high-fat diet (52% of calories as fat) with daily ethanol (4 g/kg/day, 30 days) intragastric intubation. The livers of ethanol-treated animals were characterized by fatty dystrophy. Liver triglyceride and cholesterol ester contents and the activities of serum marker enzymes, alanine aminotransferase and gamma-glutamyltransferase, were significantly increased. Ethanol enhanced phosphoinositol and sphingomyelin content in liver microsomes and lowered prostaglandin E(2) (PGE(2)) concentration in the liver. An increase in the percentage of monoenoic fatty acids and a decrease in the n-6 acid family in liver phospholipids, linoleoyl-CoA desaturase, and PGE(2) synthase activities in liver microsomes were observed in ethanol-treated rats. Treatment with UDCA improved liver morphologic characteristics, decreased triglyceride and cholesterol ester contents, increased the PGE(2) level, and normalized linoleoyl-CoA desaturase and PGE(2) synthase activities, as well as phospholipid and fatty acid patterns in the liver. The activities of the serum marker enzymes were decreased in the ethanol- and UDCA-treated group. Ursodeoxycholic acid lowered the viscosity of the microsomal membrane, as assessed by both fluorescence probe techniques and the saturated/unsaturated fatty acid ratio. We propose that the hepatoprotective effect of UDCA in alcoholic fatty liver is related to the stabilization of microsomal membranes, the prevention of a decrease in essential fatty acids and PGE(2) in the liver, and, probably, an improvement in biochemical processes controlled by PGE(2).     

Metabolic aspects of trans fatty acids

The consumption of trans isomers of unsaturated fatty acids has been associated withuntoward metabolic effects. Several clinical investigations demonstrated that trans fatty acids increase plasma LDL-cholesterol and lipoprotein (a) and reduce HDL-cholesterol concentrations. These alterations of plasma lipid profiles indicate an atherogenic effect of trans fatty acids. Both in preterm infants and in healthy children aged 1-15 years, we found blood plasma arachidonic acid (C20:4omega-6) levels and the product/substrate ratios of arachidonic acid synthesis (C20:4omega-6/C18:2omega-6) inversely correlated to the level of the principal trans fatty acid, trans octadecaenoic acid (C18:1omega-9/7, trans), which is compatible with a dose-dependent inhibition of arachidonic acid synthesis by trans fatty acids. Moreover, in premature infants trans fatty acids in blood plasma correlated inversely with birth weight in an observational study, indicating that trans fatty acids may impair early human growth. It appears desirable to limit the dietary intake of trans fatty acids. The major dietary sources of trans fatty acids are partially hydrogenated vegetable and fish oils. Refinement of the industrial technology of partial hydrogenation and appropriate food labelling may lead to a considerably decrease of human exposure to trans fatty acids.     

Effects of intramuscular injection of alpha-tocopheryl acetate on fatty acid profile in lamb liver

The effects of intramuscularly administrated vitamin E on total lipids, fatty acid profile, and lipid stability to oxidation was investigated in lamb liver. Twenty-four 5-day-old lambs were allotted to 4 groups of 6 each and given respectively 0 (control), 125, 200, 300 mg dl-alpha-tocopheryl acetate weekly from day 5 to 33. alpha-Tocopherol stored in lamb liver at the end of experiment showed linear correlation with the level of injected vitamin E. No effect on total lipids was found. A decrease in the level of liver thiobarbituric-acid reactive substances (TBARS), significantly correlated with liver alpha-tocopherol content, was found in vitamin E groups. The amount of linoleic and linolenic acids significantly increased in the vitamin E groups as compared to control group, and were correlated with the liver alpha-tocopherol content. TBARS were negatively correlated with the concentration of unsaturated fatty acids. Finally, in the liver of the treated groups, vitamin E concentrations in the range 30-50 micrograms/g showed adequate for an efficient protection from peroxidation of membrane lipids, and determined an increase in the unsaturated to saturated fatty acid ratio.     

Dietary saturated fat reduces alcoholic hepatotoxicity in rats by altering fatty acid metabolism and membrane composition

Rats fed a saturated fat diet are protected from experimentally induced alcoholic liver disease, but the molecular mechanisms underlying this phenomenon remain in dispute. We fed male Sprague-Dawley rats intragastrically by total enteral nutrition using diets with or without ethanol. In 1 control and 1 ethanol group, the dietary fat was corn oil at a level of 45% of total energy. In other groups, saturated fat [18:82 ratio of beef tallow:medium-chain triglyceride (MCT) oil] was substituted for corn oil at levels of 10, 20, and 30% of total energy, while keeping the total energy from fat at 45%. After 70 d, liver pathology, serum alanine aminotransferase (ALT), biochemical markers of oxidative stress, liver fatty acid composition, cytochrome P450 2E1 (CYP2E1) expression and activity and cytochrome P450 4A (CYP4A) expression were assessed. In rats fed the corn oil plus ethanol diet, hepatotoxicity was accompanied by oxidative stress. As dietary saturated fat content increased, all measures of hepatic pathology and oxidative stress were progressively reduced, including steatosis (P < 0.05). Thus, saturated fat protected rats from alcoholic liver disease in a dose-responsive fashion. Changes in dietary fat composition did not alter ethanol metabolism or CYP2E1 induction, but hepatic CYP4A levels increased markedly in rats fed the saturated fat diet. Dietary saturated fat also decreased liver triglyceride, PUFA, and total FFA concentrations (P < 0.05). Increases in dietary saturated fat increased liver membrane resistance to oxidative stress. In addition, reduced alcoholic steatosis was associated with reduced fatty acid synthesis in combination with increased CYP4A-catalyzed fatty acid oxidation and effects on lipid export. These findings may be important in the nutritional management and treatment of alcoholic liver disease.     

Plasma fatty acids concentrations in postoperative patients with biliary atresia.

The concentrations of plasma fatty acids in postoperative patients with biliary atresia (BA) were measured to clarify whether they had essential fatty acid deficiency. Thirty-eight fasting blood samples from 14 postoperative patients with BA were studied. All of them had the hepatic portoenterostomy without any stoma. Samples were divided into three groups on the basis of liver function. The concentrations of fatty acids in the plasma fat were measured quantitatively. Non-essential fatty acids levels were increased and omega-3 fatty acids levels were decreased with the progress of deterioration of hepatic function. Regarding omega-6 fatty acids, C18:2 and 20:4 did not show any significant difference between the three groups and the control, and only C20:3 increased with the deterioration of liver dysfunction. The ratio of C20:3 (omega-6) to C20:4 (omega-6) was increased significantly with the progress of liver dysfunction. The activity of delta-5 desaturase was suspected to be suppressed in BA patients with poor liver function. The BA patients with poor bile flow did not show any decrease of omega-6 fatty acids in the plasma, but were at risk of developing omega-3 fatty acid deficiency.     

Prevention of chemically induced diabetes mellitus in experimental animals by polyunsaturated fatty acids

Previous studies showed that essential fatty-acid deficiency, conjugated linoleic acid, and a peroxisome proliferator-activated receptor-gamma binding agent such as troglitazone can prevent the development of diabetes mellitus in experimental animals. In the present study, we observed that oral supplementation with oils rich in omega-3 eicosapentaenoic acid and docosahexaenoic acid and omega-6 gamma-linolenic acid and arachidonic acid could protect the animals against alloxan-induced diabetes mellitus. These oils rich in omega-3 and omega-6 fatty acids not only significantly attenuated chemical-induced diabetes mellitus but also restored the antioxidant status to normal range. Changes in the concentrations of different fatty acids shown by the phospholipid fractions of plasma, liver, and muscle tissues that occurred as a result of alloxan-induced diabetes mellitus also reverted to normal in these animals. Based on these results and the known mechanisms of alloxan, we suggest that omega-3 and omega-6 long-chain fatty acids can prevent chemically induced diabetes mellitus by enhancing the antioxidant status and suppressing production of cytokines.     

CYTOCHROME P-450 2E1 IN RAT LIVER, KIDNEY AND LUNG MICROSOMES AFTER CHRONIC ADMINISTRATION OF ETHANOL EITHER ORALLY OR BY INHALATION

In this study, microsomal cytochrome P-450 2E1 (CYP2E1) contentsand activities were tested in liver, kidney and lung from Wistarrats after the following treatments (1) oral administrationof a 10% ethanol solution for 4 weeks; (2) pair fed controls;(3) oral administration of a 5% acetone solution for 1 week;(4) inhalation of ethanol vapour for 4 weeks. CYP2E1 activitywas measured using chlorzoxazone as substrate and CYP2E1 contentwas measured using Western blot analysis. In addition, the cellulardistribution of CYP2E1 was studied in liver, lung and kidneyby immunohistochemistry. Basal liver CYP2E1 was 10–20times lower in lung and kidney than in liver. Inhalation wasclearly the most efficient way of inducing CYP2E1, probablydue to the continuous and high alcohol exposure. Among the organstested, lung appeared to be the tissue least sensitive to inductioneven after ethanol inhalation, suggesting the absence of localinduction. After ethanol intoxication, immunostaining was increasedin the centrilobular region of the liver, in the alveolar cellsof the lung and in the proximal convoluted tube of the kidney.The CYP2E1 activities decreased to control values in the threetissues tested, within 24 h after cessation of intoxication.     

The omega-6/omega-3 fatty acid ratio, genetic variation, and cardiovascular disease

A high omega-6/omega-3 ratio, as is found in today's Western diets, promotes the pathogenesis of many chronic diseases, including cardiovascular disease. Increased dietary intake of linoleic acid (LA) leads to oxidation of low-density lipoprotein (LDL), platelet aggregation, and interferes with the incorporation of essential fatty acids (EFA) in cell membrane phopholipids. Both omega-6 and omega-3 fatty acids influence gene expression. Omega-3 fatty acids have strong anti-inflammatory effects, suppress interleukin 1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF alpha) and interleukin-6 (IL-6), whereas omega-6 fatty acids tend to be pro-inflammatory. Because inflammation is at the base of many chronic diseases, including coronary heart disease, dietary intake of omega-3 fatty acids plays an important role in the manifestation of disease, particularly in persons with genetic variation, as for example in individuals with genetic variants at the 5-lipoxygenase (5-LO). Increased dietary arachidonic acid (AA) significantly enhances the apparent atherogenic effect of genotype, whereas increased dietary intake of omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) blunts this effect. The diet-gene interaction further suggests that dietary omega-6 fatty acids promote, whereas marine omega-3 fatty acids EPA and DHA inhibit leukotriene-mediated inflammation that leads to atherosclerosis in this subpopulation.     

Long-chain polyunsaturated fatty acids and chemically induced diabetes mellitus: effect of omega-6 fatty acids

OBJECTIVE: We previously showed that prior oral supplementation of oils rich in omega-3, eicosapentaenoic acid and docosahexaenoic acid, and omega-6, gamma-linolenic acid and arachidonic acid, can prevent the development of alloxan-induced diabetes mellitus in experimental animals. But the effect of individual fatty acids on chemically induced diabetes mellitus is not known. We report the results of our studies with omega-6 fatty acids. METHODS: Alloxan-induced in vitro cytotoxicity and apoptosis in an insulin-secreting rat insulinoma cell line, RIN, was prevented by prior exposure of these cells to linoleic acid, gamma-linolenic acid, and arachidonic acid (AA) but not to dihomo-gamma-linolenic acid. Cyclo-oxygenase and lipoxygenase inhibitors did not block this protective action of AA. Prior oral supplementation with gamma-linolenic acid and pre- and simultaneous treatments with AA prevented alloxan-induced diabetes mellitus. RESULTS: Even though pretreatment with linoleic acid and dihomo-gamma-linolenic acid and simultaneous treatment with linoleic acid, gamma-linolenic acid, and dihomo-gamma-linolenic acid did not prevent the development of diabetes mellitus, the severity of diabetes was much less. The saturated fatty acid stearic acid and the monounsaturated fatty acid oleic acid were ineffective in preventing alloxan-induced diabetes mellitus. gamma-Linolenic acid and AA not only attenuated chemically induced diabetes mellitus but also restored the antioxidant status to normal range in various tissues. Changes in the concentrations of various fatty acids of the phospholipid fraction of plasma that occurred as a result of alloxan-induced diabetes mellitus also reverted to normal in the AA-treated animals. CONCLUSIONS: These results suggest that polyunsaturated fatty acids can prevent chemically induced diabetes in experimental animals and attenuate the oxidant stress that occurs in diabetes mellitus.     

Modulation of human immune and inflammatory responses by dietary fatty acids.

I review the effects of the amount and composition of dietary fat on indices of human immune and inflammatory responses. A reduction in the amount of fat intake enhanced several indices of immune response, including lymphocyte proliferation, natural-killer-cell activity, cytokine production, and delayed-type hypersensitivity. When total fat intake was held constant, an increase in the intake of linoleic acid (18:2 omega-6) or arachidonic acid (20:4 omega-6) by healthy human volunteers did not inhibit many indices of immune response tested but did increase the production of inflammatory eicosanoids (prostaglandin E2 and leukotriene B4). Supplementation of human diets with omega-3 fatty acids reduced several aspects of neutrophil, monocyte, and lymphocyte functions, including the production of inflammatory mediators. Most of the studies have indicated reductions in these functions, with a minimum of 1.2 g/d of supplementation with eicosapentaenoic acid and docosahexaenoic acid for 6 wk. However, other studies concomitantly supplementing with 205 mg/d of vitamin E did not find inhibition of immune-cell functions, even with larger amounts and longer durations of supplementation with these fatty acids. One study reported that supplementation with docosahexaenoic acid selectively inhibits inflammatory responses without inhibiting T- and B-cell functions. Despite some discrepancies, fish oils have been used successfully in the management of several inflammatory and autoimmune diseases. The potential for the use of fish oils in the management of these diseases is tremendous, even though further studies are needed to establish safe and adequate intake levels of omega-3 fatty acids.     

Suppressive effect of dietary unsaturated fatty acids on alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase, a key enzyme of tryptophan-niacin metabolism in rat liver

The effect of dietary fat on the activity of rat liver alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase [EC 4.1.1.45] (ACMSD), a key enzyme of tryptophan-niacin metabolism, was investigated. When high-fat diet was given to rats for a week, the activity of ACMSD in the liver was extremely suppressed, but not in the kidney. The liver enzyme activity was correspondingly and constantly reduced half by increasing soybean oil in the diet in 4% stages. The potency of suppression of the enzyme activity in liver was found to be different between soybean oil and lard in diets. Among various dietary fatty acids, saturated fatty acid was observed to be less effective. Unsaturated fatty acids, however, were shown to be suppressive and polyunsaturated fatty acid such as linoleic and linolenic acids more effective than oleic acid in suppressing liver ACMSD activity. These suppressive effects of dietary unsaturated fatty acids on the liver ACMSD activity were not considered to be caused by their direct effect on the enzyme protein. Moreover, neither inhibitor nor activator was thought to be involved in the alteration of the enzyme activity.     

Iron and CYP2E1-dependent oxidative stress and toxicity.

Iron plays a critical role in catalyzing the formation of potent oxidants. Increases in iron content enhance oxidative stress, whereas removal of iron deceases such stress. An association between iron and alcoholic liver injury has been proposed. The ability of iron to modulate the biochemical and toxicologic actions of cytochrome P450 2E1 (CYP2E1) has been evaluated by using isolated microsomes and intact liver cells. The ability of different iron complexes to stimulate microsomal lipid peroxidation and hydroxyl radical production during reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)- and reduced form of nicotinamide adenine dinucleotide (NADH)-dependent electron transfer has been characterized. Certain iron complexes have been shown to be effective in promoting lipid peroxidation; others are better catalysts of hydroxyl radical production as a complex pattern has been found. Reactive oxygen production, lipid peroxidation, and interaction with iron chelates have been shown to be enhanced with microsomes isolated from ethanol-treated rats with elevated levels of CYP2E1. This increase was prevented by anti-CYP2E1 immunoglobulin (Ig)G or chemical inhibitors of CYP2E1. Thus, in the presence of iron complexes, microsomes enriched in CYP2E1 are especially reactive in generation of reactive oxygen species. To assess the toxicologic significance of this iron-CYP2E1 interaction, iron (ferric-nitrilotriacetate) was added to HepG2 cells, which were engineered to express the human CYP2E1. Ferric-nitrilotriacetate produced a greater toxicity in the CYP2E1-expressing HepG2 cells than that in control HepG2 cells. This enhanced, synergistic toxicity was blocked by antioxidants and inhibitors of CYP2E1. Mitochondrial membrane potential and ATP levels were decreased, and damage to the mitochondria played a critical role in the CYP2E1-plus-iron-dependent toxicity. These results support the suggestion that low concentrations of iron and polyunsaturated fatty acids can act as priming or sensitizing factors for CYP2E1-induced injury in HepG2 cells and hepatocytes. Such interactions may play a role in alcohol-induced liver injury.     

Effects of dietary linoelaidate on fatty acid composition and phospholipase activity in rat liver microsomes

The activity of calcium-stimulated microsomal phospholipase towards endogenous membrane bound phospholipids was monitored by analyzing the fatty acids released using gas chromatography. Activation by calcium (10 mM) resulted in the release of 2.1 nmoles fatty acid/10 mg microsomal protein/hr. The pattern of release of particular fatty acids reflected their concentration in the microsomal phospholipids.The effects of feeding 10 and 50% dietary linoelaidate (t,t18:2) and hydrogenated tallow (i.e., essential fatty acid deficient) on microsomal fatty acids and phospholipase activity was determined. The levels of t,t18:2 in the microsomal phospholipids increased with dietary intake and duration of feeding and attained 8.4% of total fatty acids after 11 weeks. There was a concomitant increase in linoleic acid and a decrease in arachidonic acid levels in microsomal lipids. The microsomal phospholipids of animals receiving exclusively hydrogenated tallow contained only trace amounts of linoleic and arachidonic acid after four weeks. The amount of eicosatrienoic acid increased dramatically in these animals. Palmitoleic and oleic acid also increased. The calcium-stimulated phospholipase activity in liver microsomes was unchanged by alterations in the fatty acid composition of the phospholipids.