The effect of anoxia on lipid metabolism in isolated adult rat cardiac myocytes

In ischemic myocardium abnormal lipid metabolism results in accumulation of compounds that are deleterious to membrane structural integrity and membrane dependent functions. In this study isolated adult rat ventricular myocytes were used to investigate anoxia-induced alterations in cellular lipid composition and metabolism. Myocyte phospholipid content declined 19% on average during 60 min anoxia and intracellular arachidonic acid increased 3-fold, without affecting myocyte ATP content. Anaerobic incubation in the absence of glucose depleted cellular ATP to 2 nmol/mg protein, elicited a 23% decrease in phospholipids, and reduced triacylglycerol content by 51%. Intracellular levels of C16-C22 fatty acids were significantly elevated, especially palmitic and arachidonic acids. Myocytes presented with 0.08 mM [1-14C]-palmitic or arachidonic acid acylated 85% (25-26 nmol/mg) of the fatty acid taken up into triacylglycerols. Anoxia decreased this esterification by 46-60%. Formation of [14C]-CO2 was also depressed 70-90% by anaerobiosis. The results demonstrate that anoxia stimulates degradation of complex lipids, with a concomitant increase in non-esterified fatty acids, especially arachidonic acid.     

Selective uptake of [3H]arachidonic acid into the dense tubular system of human platelets

We have used quantitative electron microscopic autoradiography to characterize the subcellular distribution of arachidonoyl phospholipids following brief (5 minutes) exposure of unstimulated human platelets to [3H]arachidonic acid. Labeled arachidonate was taken up rapidly and incorporated into phospholipids. Phospholipid radioactivity was preserved and spatially fixed during tissue processing for electron microscopy. Analysis of autoradiographs showed that following a brief exposure to 750 nmol/L [3H]arachidonate, there is selective labeling of an internal membrane compartment composed of the dense tubular system and the open canalicular system. The plasma membrane, platelet granules, and nonmembranous cytoplasm were not labeled. Since the open canalicular system is continuous with the plasma membrane and since phospholipids in continuous membranes are freely diffusible, our observations indicate that [3H]arachidonate was incorporated into phospholipids within the dense tubular system and not the open canalicular system. Thus, the dense tubular system, known to contain cyclooxygenase activity, incorporates arachidonate selectively following brief exposure to this fatty acid, presumably to concentrate it in proximity to enzymes for icosanoid synthesis.     

Effects of Arachidonic Acid on Hepatic Lipids in Ethanol-Fed Rats

The effects of arachidonic acid supplementation on rats fed ethanol employing an ad libitum schedule have been reported to be different from those observed when rats are fed in more limiting, matched fashion. To reexamine this issue, rats were fed unrestricted amounts of a diet in which 36% of the energy was provided by either ethanol or isocaloric amounts of carbohydrate. In half the animals, 7% of fat consisted of arachidonic acid. Despite earlier reports to the contrary, arachidonic acid had no effect on weight gain and did not attenuate the ethanol-induced fatty liver. Arachidonate supplementation tended to increase hepatic total lipids and triacylglycerols, and to potentiate the ethanol-induced elevation of cholesterol esters. Our present results are consistent with those previously reported using pair-feeding techniques in which dietary intakes are somewhat limited. Thus, regardless of the feeding technique employed, relative arachidonic deficiency cannot be invoked to explain the lipid accumulation observed after chronic ethanol consumption.     

Effects of L-leucine on palmitate metabolism and insulin release by isolated islets of fed and starved rats

The occurrence of lipid metabolic changes associated with L-leucine (10 mM) stimulation of insulin release was investigated in isolated islets from either fed or starved rats. L-Leucine-stimulated secretion was potentiated by 3 mM glucose and/or 0.5 mM palmitate and was unaffected by 48 h of starvation. Islet palmitate oxidation showed a maximum rate at 3 mM glucose, and starvation increased it almost 2-fold. Regardless of the nutritional state, L-leucine strongly reduced the oxidation of palmitate and increased its incorporation into islet triacylglycerols and phospholipids at 3 mM glucose. This shift of fatty acid metabolism toward esterification might play a role in the mechanism of potentiation of the islet secretory response to L-leucine by glucose and palmitate.     

Phospholipid metabolism of hypothermically stored rat hepatocytes.

Isolated rat hepatocytes were suspended and stored in either Liebovitz-15 medium (37 degrees C or 4 degrees C) or University of Wisconsin (UW) solution (4 degrees C) containing [(3)H] arachidonic acid (AA). At varying times, membrane phospholipids were separated by thin layer chromatography. AA labeled phospholipids similarly at both 4 degrees C and 37 degrees C. Analysis of the ratios of [(3)H] AA and [(14)C] glycerol incorporated into phosphatidic acid or other phospholipids in dual-labeled cells indicated that the deacylation/reacylation cycle was the major route of AA incorporation at hypothermia. This was supported by showing that blocking phospholipase A(2) (PLA(2)) activity by trifluoperazine suppressed AA incorporation into phospholipids. PLA(2) activity, measured by determining the release of AA, was slow during 48-hour cold storage, but increased significantly when ATP was depleted by inhibition of mitochondria and glycolysis. In the whole rat liver, there was no significant loss of phospholipids during 48-hour storage (total phospholipids [micromol phosphorus/L/mg] : 0.197 +/-. 001 at 0 hours) unless energy blockers were used (0.155 +/-.005 at 48 hours) or glycogen depleted by fasting the rat (0.167 +/-.001 at 48 hours). This study shows that a net PLA(2) stimulated hydrolysis of phospholipids is seen only when ATP is depleted and its generation from anaerobic glycolysis inhibited. Thus, PLA(2) hydrolysis of phospholipids is not a significant cause of liver cell injury during cold storage when livers are obtained in optimal condition. However, conditions affecting the generation of ATP during cold storage could alter PLA(2) leading to membrane damage.     

Ethanol toxicity in pancreatic acinar cells: mediation by nonoxidative fatty acid metabolites

Ethanol causes pancreatic damage by an unknown mechanism. Previously, we demonstrated that a sustained rise of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) causes pancreatic acinar cell injury. Here we have investigated the effects of ethanol and its metabolites on Ca(2+) signaling in pancreatic acinar cells. Most cells exposed to ethanol (up to 850 mM) showed little or no increase in [Ca(2+)](i) (and never at concentrations <50 mM). During sustained exposure to 850 mM ethanol, acetylcholine (ACh) evoked a normal [Ca(2+)](i) elevation and following ACh removal there was a normal and rapid recovery to a low resting level. The oxidative metabolite acetaldehyde (up to 5 mM) had no effect, whereas the nonoxidative unsaturated metabolite palmitoleic acid ethyl ester (10-100 microM, added on top of 850 mM ethanol) induced sustained, concentration-dependent increases in [Ca(2+)](i) that were acutely dependent on external Ca(2+) and caused cell death. These actions were shared by the unsaturated metabolite arachidonic acid ethyl ester, the saturated equivalents palmitic and arachidic acid ethyl esters, and the fatty acid palmitoleic acid. In the absence of external Ca(2+), releasing all Ca(2+) from the endoplasmic reticulum by ACh (10 microM) or the specific Ca(2+) pump inhibitor thapsigargin (2 microM) prevented such Ca(2+) signal generation. We conclude that nonoxidative fatty acid metabolites, rather than ethanol itself, are responsible for the marked elevations of [Ca(2+)](i) that mediate toxicity in the pancreatic acinar cell and that these compounds act primarily by releasing Ca(2+) from the endoplasmic reticulum.     

Defining erythrocyte internal labeling by phosphorylation.

When erythrocytes are incubated with 32Pi, incorporation of label into phosphoproteins is a gradual process, increasing for at least 2 hours. Membrane phospholipids also are labeled. Exogenous protein kinase substrates are unlabeled in these incubations. This suggests that labeling by 32Pi occurs into polypeptides inside the erythrocytes. When erythrocytes are incubated with [gamma-32P]ATP and active protein kinase, membrane polypeptides are not labeled. Only exogenously added protein kinase substrates and the regulatory subunit of protein kinase (and its contaminants) are labeled. This suggests that labeling from [gamma-32P]ATP and active protein kinase occurs in the compartment outside the erythrocytes. Apyrase (EC 3.6.1.5) eliminates such labeling, demonstrating that it was occurring in the compartment external to the erythrocytes. However, in incubations of cells with 32Pi, apyrase has no effect on the incorporation into membrane polypeptides and phospholipids, demonstrating that this labeling occurs on the inside of the membrane. Thus, additions of apyrase to intact particles incubated with protein kinase substrates and 32Pi provides a method for identifying internally exposed polypeptides in the plasma membranes of a variety of systems.     

Oleate metabolism and endogenous triacylglycerol hydrolysis in isolated hepatocytes from rats fed a high-fat diet

In isolated hepatocytes of fat-fed rats, as compared to control fed animals, the cellular uptake of [1-14C] oleate and its oxidation to CO2 were similar but the incorporation of the label into water-soluble products (mainly ketone bodies) was increased by 36.6% whereas its esterification to triacylglycerols and phospholipids decreased by 36%. While endogenous ketogenesis was slightly but not significantly increased, ketone body synthesis from both 2 mM octanoate and 0.7 mM oleate was stimulated two fold. Thus, in the fatfed rats the oxidative pathway is clearly activated whereas long chain fatty acids are preferentially channelled into the oxidation pathway at the expense of esterification. Yet, hepatocyte triacylglycerol content was 3-fold higher after fat-feeding. In this regard, lysosomal triacylglycerol lipase (EC 3.1.1.3) activity, in homogenates of hepatocytes was decreased by 32% (p less than 0.01). This findings suggest a lower breakdown of endogenous triacylglycerols, which, taken together with decreased secretion of VLDL lipoprotein triacylglycerol (Kalopissis et al. Biochem. J. 198: 373, 1981) and an in vivo increased fatty acid influx to the liver may contribute to the accumulation of lipids in the livers of fat-fed rats.     

Effect of Dietary Ethanol and Cholesterol on Phospholipid Composition of Hepatic Mitochondria and Microsomes from the Monkey, Macaca nemestrina

Monkeys (Macaca nemestrina) were divided into four groups, and each group was fed a particular diet. The variables in the diets were as follows: diet A, 0.3 mg cholesterol/kcal nutrient; diet B, 1.0 mg cholesterol/kcal nutrient; diet C, 0.3 mg cholesterol/kcal nutrient, ethanol (36% of calories); diet D, 1.0 mg cholesterol/ kcal nutrient, ethanol (36% of calories). Monkeys on the diets containing ethanol developed fatty liver. Mitochondria and microsomes isolated from these livers demonstrated ethanol-elicited alterations in metabolic functions as is described in the preceding paper.¹ Accompanying these changes in metabolic activities were alterations in organelle phospholipids that were influenced by both dietary ethanol and cholesterol. The changes that could be attributed to ethanol were as follows. Phosphatidyl ethanolamine was decreased in microsomes and increased in mitochondria; the sphingomyelin content in microsomes was increased significantly. The levels of stearic and arachidonic acid were elevated, and palmitic and oleic acid decreased, in phospholipids from both mitochondria and microsomes. Cholesterol influenced the fatty acid composition of several phospholipids, usually in a direction opposite to those alterations attributed to ethanol. Cholesterol feeding increased levels of palmitic and oleic acid and decreased amounts of stearic, linoleic, and arachidonic acid in several phospholipids. The significant ethanol- and cholesterol-elicited alterations observed in this study suggest the possibility that the changes in metabolic functions in mitochondria and microsomes are controlled, at least in part, by alterations in the phospholipid compositions of these organmicrosomes are controlled, at least In part, by alterations in the phospholipid compositions of these organelles.     

Ischemia and reperfusion induced multilamellar vesicles in isolated rabbit hearts: time correlation between morphometric data and metabolic alterations

In normoxic hearts a limited number of multilamellar vesicles was found in both endothelial cells and myocytes. The total number of multilamellar vesicles observed in myocytes, particularly those extruded from mitochondria, significantly increased in hearts rendered ischemic for at least 60 mins. The number of multilamellar vesicles extruded from sarcolemma was increased in hearts reperfused after this period of ischemia. The number of multilamellar vesicles in or adjacent to lipid droplets was independent of the duration of ischemia. Multilamellar vesicles were similar in size and periodicity of the lamellae. It is proposed that the number of multilamellar vesicles can be used to quantitate ischemic membrane injury. The formation of multilamellar vesicles was significantly related in time to (a) the accumulation of arachidonic acid and total fatty acids; (b) a decrease in the tissue content of ATP and (c) the release of lactate dehydrogenase (LDH). No significant alterations in the total tissue content of triacylglycerols and phospholipids were detected. The amount of arachidonic acid accumulated in the hearts reflects the degradation of only a minor fraction of the phospholipid pool. Assuming a close relationship between phospholipid degradation, induction of multilamellar vesicles and loss of cellular integrity, the present findings might indicate that the loss of a small part of phospholipids might have serious pathophysiological consequences, as indicated by the morphological changes in cellular membranes and the release of cytoplasmic macromolecules.     

The effect of thrombin on the uptake and transformation of arachidonic acid by human platelets

Washed human platelets take up arachidonic acid from plasma and incorporate the fatty acid into the major classes of complex lipids. Thrombin impairs net incorporation. It activates endogenous phospholipases which liberate arachidonic acid from phospholipids. As a consequence of thrombin induced aggregation platelets release arachidonic acid intermediates formed by the action of platelet fatty acid cyclooxygenase and by platelet fatty acid lipoxygenase. Cyclooxygenase, but not lipoxygenase, is inhibited by aspirin and indomethicin. Analysis of the pathways of arachidonic acid metabolism may furnish new insight into platelet function and into disorders of primary hemostasis.     

Non-oxidative metabolism of ethanol by rat pancreatic acini.

BACKGROUND: The pathogenesis of alcoholic pancreatitis may involve the metabolism of ethanol (via oxidative and non-oxidative pathways) within the pancreas. The aims of this study were to determine the rate of non-oxidative metabolism in isolated rat pancreatic acini and to compare this to the rate of ethanol oxidation. METHODS: Pancreatic acini were isolated from male Sprague-Dawley rats and incubated with (14)C-ethanol. Radiolabelled fatty acid ethyl esters (non-oxidative metabolites) were isolated from lipid extracts by thin-layer chromatography. Radiolabelled acetate (oxidative metabolite) was isolated from the incubation medium by ion-exchange chromatography. RESULTS: Non-oxidative metabolism by isolated pancreatic acini was demonstrated. At 50 and 100 mmol/l ethanol, fatty acid ethyl ester concentrations were 49.6 +/- 13.3 and 199 +/- 93 micromol/l, respectively. These levels have previously been shown to result in tissue injury. Non-oxidative metabolism was increased 9-fold by addition of oleic acid and inhibited by the lipase inhibitor, tetrahydrolipstatin, by 91.05 +/- 1.99%. The rate of oxidative metabolism was 21-fold higher than that of non-oxidative metabolism. CONCLUSIONS: Intact pancreatic cells metabolize ethanol by the non-oxidative pathway, generating fatty acid ethyl esters at a rate sufficient to cause pancreatic damage. Oxidative metabolism of ethanol occurs at a much higher rate and may also play a role in pancreatitis.     

Effects of Chronic Ethanol on Enzymes Regulating Sialylation and Desialylation of Transferrin in Rats

Transferrin is a N-glycosylated glycoprotein and plays an important role in iron transport from sites of absorption and storage to sites of utilization. Chronic ethanol alters the normal microheterogeneity pattern of transferrin as a consequence of changes in the sialic acid content. However the underlying basis of this change in sialic acid contents of transferrin in alcohol abuse remains unclear. We have undertaken this study in order to investigate the effects of chronic ethanol in rats with respect to the hepatic rate of (i) transferrin synthesis based on labeled leucine incorporation, (ii) the incorporation of labeled N-acetyl mannosamine (NAM) into sialic acid residues of transferrin, and (iii) roles of specific sialyltransferase and sialidase at hepatic subcellular level. The results showed no significant difference in the incorporation of labeled leucine into transferrin at all levels between the control and ethanol group, whereas the incorporation of NAM into transferrin was significantly decreased by 84% (p < 0.001) both at the whole cell and Golgi level. Thus, the incorporation of labeled NAM relative to the incorporation of labeled leucine into hepatic transferrin was significantly decreased by 86% (p < 0.001) in chronic ethanol-treated animals as compared with the controls both at the whole cell and Golgi levels. These data are further supported by our finding of concomitant decrease in the activity of β-galactoside α2,6-sialyltransferase by 58% (p < 0.01) in ethanol-treated rats as compared with control animals. In contrast, both the plasma membrane and plasma sialidase activities were increased by 95% (p < 0.01) and 85% (p < 0.01), respectively, in ethanol-fed rats as compared with their respective controls. We conclude that decreased activity of sialyltransferase and increased activity of sialidase sequentially at the plasma membrane and plasma compartment may be responsible for decreased incorporation of sialic acid residues in serum transferrin molecules after chronic ethanol treatment.     

Protective role of intracoronary fatty acid binding protein in ischemic and reperfused myocardium

In this study, fatty acid binding protein was used to protect an ischemic heart from reperfusion injury. Isolated rat heart was preperfused in the presence of 1.4 microM liposome-bound fatty acid binding protein for 15 minutes, followed by 30 minutes of ischemia and 30 minutes of reperfusion. Our results indicated better preservation of myocardial high-energy phosphate compounds (including ATP and creatine phosphate), reduced creatine kinase and lactate dehydrogenase release from the heart, and improved coronary flow in hearts treated with fatty acid binding protein compared with untreated controls. Fatty acid binding protein enhanced reacylation of arachidonic acid into phospholipids, thereby preserving membrane phospholipids and reducing free fatty acid contents during ischemia and reperfusion. In addition, fatty acid binding protein-bound long-chain free fatty acids and their thioesters as well as carnitine esters were increased in the cytosolic compartment of the heart. These results suggest that fatty acid binding protein may be used as a possible therapeutic agent to improve myocardial function during reperfusion of ischemic heart.     

Membranes and phospholipids of liver mitochondria from chronic alcoholic rats are resistant to membrane disordering by alcohol.

Using the spin probe 5-doxylstearic acid, we studied the structural perturbations of rat liver mitochondrial membranes produced by exposure to ethanol in vitro and by chronic ethanol feeding. The addition of ethanol in vitro to mitochondria from control animals appears to "fluidize" the membranes, as evidenced by a pronounced decrease in the order parameter. By contrast, in membranes from rats fed ethanol chronically, there was no effect on the order parameter. This resistance of the mitochondrial membranes from chronically intoxicated animals to the fluidizing effect of ethanol probably results from a change in the composition of the phospholipids, because the same differential response to ethanol was observed upon using vesicles of mitochondrial phospholipids extracted from control and chronically treated rats. In the presence of 0.025--0.1 M ethanol, a range that prevails in the blood of chronic alcoholics, the order parameter of mitochondrial membranes from rats fed ethanol was comparable to that of control membranes without ethanol in vitro. Analysis of extracted mitochondrial phospholipids showed that the cardiolipin from ethanol-fed animals had fatty acyl residues that are more saturated than those of controls. These findings point to the underlying molecular mechanism of our previous observation that mitochondria from chronic alcoholic rats are more resistant to uncoupling by ethanol at physiological temperature [Rottenberg, H., Robertson, D. E. & Rubin, E. (1980) Lab. Invest. 42, 318--326]. We suggest that an adaptive change in the phospholipid composition leads to structural alterations, which result in increased resistance to disruption of mitochondrial membranes by ethanol. These changes in lipid composition and structure may explain many, if not all, of the mitochondrial abnormalities that have been previously reported to result from chronic ethanol intoxication.     

Metabolic labeling and direct imaging of choline phospholipids in vivo

Choline (Cho)-containing phospholipids are the most abundant phospholipids in cellular membranes and play fundamental structural as well as regulatory roles in cell metabolism and signaling. Although much is known about the biochemistry and metabolism of Cho phospholipids, their cell biology has remained obscure, due to the lack of methods for their direct microscopic visualization in cells. We developed a simple and robust method to label Cho phospholipids in vivo, based on the metabolic incorporation of the Cho analog propargylcholine (propargyl-Cho) into phospholipids. The resulting propargyl-labeled phospholipid molecules can be visualized with high sensitivity and spatial resolution in cells via a Cu(I)-catalyzed cycloaddition reaction between the terminal alkyne group of propargyl-Cho and a labeled azide. Total lipid analysis of labeled cells shows strong incorporation of propargyl-Cho into all classes of Cho phospholipids; furthermore, the fatty acid composition of propargyl-Cho-labeled phospholipids is very similar to that of normal Cho phospholipids. We demonstrate the use of propargyl-Cho in cultured cells, by imaging phospholipid synthesis, turnover, and subcellular localization by both fluorescence and electron microscopy. Finally, we use propargyl-Cho to assay microscopically phospholipid synthesis in vivo in mouse tissues.     

Myocardial metabolites of ethanol

Because of the importance of alcohol-induced heart muscle disease and the obscurity of its pathogenesis, this study was undertaken to determine whether fatty acid ethyl esters, myocardial metabolites of ethanol recently described in our laboratory to be synthesized in cell-free extracts of rabbit myocardium, accumulate in hearts of human subjects exposed to ethanol in vivo. Lipid extracts were prepared from left ventricular samples obtained at necropsy from six subjects who had been exposed to ethanol acutely or chronically. Fatty acid ethyl esters were present in each extract in concentrations ranging from 9 to 115 microM. In contrast, they were consistently absent from analogous samples obtained from hearts of abstainers (n = 5). In parallel studies in experimental animals, we found that fatty acid ethyl esters are formed not only in the heart but also in the pancreas and liver--targets of injury associated with chronic alcohol abuse. These results demonstrate the presence in human myocardium of a novel metabolite of ethanol that potentially may serve as a marker for exposure to alcohol and that could be relevant to the pathophysiology of excessive alcohol consumption leading to cardiac abnormalities.     

The Metabolism of Ethyl Esters of Fatty Acids in Adipose Tissue of Rats Chronically Exposed to Ethanol

The concentration of ethyl esters of fatty acids as well as the activity of the enzyme synthesizing these esters (fatty acid ethyl ester synthase) were determined in adipose tissue of rats ingesting ethanol (9-16 g/kg body weight/day) for different periods of time. After 10 and 17 weeks of ethanol exposure about 300 nmol of ethyl esters of oleic, palmitic, stearic, and linoleic acids were found per gram adipose tissue. The ethyl esters disappeared after 1 week of abstinence. Closer analyses, using radioactive ethanol, revealed a half-life of the esters of less than 24 hr. The bulk of the esters was found in a membrane preparation of isolated adipocytes. Hormone-sensitive lipase hydrolyzed emulsified ethyl oleate as efficiently as that of trioleoylglycerol, but in mixed ethyl oleate/trioleoyl glycerol particles the hydrolysis of ethyl oleate was slower, suggesting a decreased accessibility. Synthase activity was found in adipose tissue from rats not exposed to ethanol. It doubled after 10 and 17 weeks of ethanol and decreased with a half-life of at least a week after abstinence. It was concluded that ethyl esters of fatty acids are formed in rat adipose tissue as previously shown in other tissues. They seem to be stored mainly in membranous parts of the adipocytes. Synthase activity is induced by ethanol. The elevated activity has a longer half-life, and may be useful as an indicator of alcohol abuse.     

The Human Blood Platelet as a Model for Studying Interactions of Ethanol with Membrane Lipids

The effect of ethanol on human blood platelet aggregation is generally inhibitory, but aggregation caused by arachidonic acid is either unaffected or potentiated by ethanol. Of the other aggregatory agents tested, the calcium ionophore A23187, collagen, and thrombin were most inhibited by ethanol. These results suggest that in the case of collagen and ionophore A23187 ethanol may act to inhibit aggregation at some point between the rise in cytosolic calcium and the cleavage of membrane phospholipids associated with the platelet release reaction. A similar spectrum of inhibition was produced by the incorporation of unsaturated fats into the platelet or by reduction of divalent cations by addition of EDTA to the external medium. Platelets in which unsaturated fats were incorporated were less susceptible to inhibition by ethanol than those into which saturated fats had been incorporated.     

Phosphatidylcholine biosynthesis in myocardial ischaemia

In this study the effect of myocardial ischaemia was evaluated on two aspects of phospholipid metabolism: (i) the de novo synthesis of myocardial phospholipids, as indicated by the incorporation of (methyl-3H) choline and (ii) the incorporation of radiolabelled long chain fatty acids into tissue phospholipids. Two models of ischaemia were used namely normothermic ischaemic arrest and hypoxic, low-flow perfusion of the isolated rat heart. The results showed that within 10 min, hypoxic low-flow perfusion significantly inhibited the incorporation rate of (methyl-3H) choline into tissue phospholipids. Since the tissue choline content remained unaltered under these conditions, the results suggested that the de novo synthesis of phosphatidylcholine is very susceptible to ischaemic damage. Inhibition of (methyl-3H) choline incorporation into tissue phospholipids appeared to be due to both a reduction in choline uptake and specific inhibition of the CDP pathway. Perfusion with glucose (10 mM) as substrate completely abolished the ischaemia-induced reduction in (methyl-3H) choline incorporation, indicating that glycolytically produced ATP played an important role in phosphatidylcholine biosynthesis. In contrast to these results, myocardial ischaemia stimulated the incorporation of long-chain saturated and unsaturated fatty acids into tissue phospholipids. In summary, the results obtained showed that myocardial ischaemia profoundly affected phospholipid metabolism which, in turn, might contribute to membrane damage.