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Recent studies have shown that, in addition to free steroid hormones, those bound to albumin in plasma may also be available to tissues. In this report, the effects of free fatty acids (FFA) on the binding of steroids to albumin were compared for the cases of bovine serum albumin (BSA) and human serum albumin (HSA). The apparent association constant, Ka, was estimated from the changes in the equilibrium partition coefficient of steroids between the aqueous/hexane phases caused by the addition of albumin to the aqueous phase. In the case of BSA, Ka for progesterone and testosterone increased upon binding of FFA (myristic, palmitic and stearic acid) to BSA and the maximum value of Ka for these steroids could be attained by 3--4 mol of FFA bound per mol BSA. Furthermore, the elution profiles of gel-filtration chromatography clearly showed that progesterone and testosterone are easily liberated from the steroid/BSA complexes and that FFA potentiates the binding of these steroids to BSA. In the case of HSA, the binding affinities of progesterone and testosterone were not greatly affected by bound FFA. On the other hand, the affinities of ethynylestradiol to both BSA and HSA were unaffected below 4 mol of FFA binding per mol.

Production of platelet-activating factor (PAF) during opsonized zymosan stimulation of human polymorphonuclear leukocytes is dependent on the concentration of extracellular albumin and on the presence of exogenous fatty acids. Fatty acid-free albumin caused a concentration-dependent increase in PAF synthesis up to 5% albumin concentrations (w/v) where the amount of PAF produced was three- to four-fold higher than in controls containing no albumin. The addition of free fatty acids, particularly arachidonic acid and palmitic acid, to 5% fatty acid-free albumin media caused a concentration-dependent decrease in PAF synthesis. A 50% inhibition of PAF synthesis was observed at an arachidonic acid concentration of 120 microM and at a palmitic acid concentration of 100 microM. The inhibition of PAF production by palmitic acid was also dependent on the concentration of extracellular albumin. In 0.5% fatty acid-free albumin media, a palmitic acid concentration of 40 microM produced a 50% inhibition in PAF synthesis. The addition of palmitic acid did not affect the release of endogenous arachidonic acid during stimulation. In contrast, the addition of stearic acid up to 120 microM in 5% fatty acid-free albumin media had no effect on PAF production. The different inhibitory effects of palmitic acid and stearic acid on PAF production may be related to differences in intracellular utilization of these two fatty acids during cell stimulation.

The effect of divalent cation ionophore, A23187, on the incorporation of [1-14C]palmitic acid, [1-14C]linoleic acid and [U-14C]glycerol into glycerolipids of polymorphonulcear leukocytes was examined. Ionophore A23187 stimulated the labeling of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, and diacylglycerol by both labeled fatty acids and glycerol. [1-14C]Palmitic acid and [1-14C]linoleic acid incorporation into phosphatidylcholine and triacylglycerol was reduced by the presence of the ionophore in the incubation medium, while [U-14C]glycerol labeling of these lipids was not significantly changed under identical conditions. These data reflect that the acylation of sn-glycerol 3-phosphate is activated, and the acylations of lysophosphatidyl-choline and endogenous diacylglycerol are inhibited in cells incubated with ionophore A23187. External calcium was not required for the ionophore effect on the incorporation of labeled fatty acids and glycerol. It is suggested that the ionophore alters the metabolism of the fatty acid and glycerol moieties of glycerolipids by changing the distribution of intracellular calcium of leukocytes.

Perturbation of the fatty acid composition of human lymphocytes in vitro was investigated by addition of linoleic acid complexed to bovine serum albumin (BSA-LA) and by mitogenic stimulation with phytohaemagglutinin (PHA). BSA-LA resulted in a 45% increase in linoleic acid in phosphatidylethanolamine (PE) and over 100% in phosphatidylcholine (PC) in peripheral blood cells. Supplementation with BSA-LA in PHA-stimulated lymphocytes produced even greater changes: 100% increase in linoleic acid content for PE and over 300% for PC. There was a large decrease in oleic acid: 40% for PE and almost 100% in PC. Significant decreases in arachidonic acid occurred in both phospholipid fractions. PHA alone also altered membrane phospholipid fatty acid composition, with reductions in palmitic, stearic and linoleic acid for PE and increases in oleic acid and arachidonic acid (almost 100%). For PC, there were large decreases in stearic (40%), linoleic (30%) and arachidonic (40%) acids, together with an increase in oleic acid (65%). Cells supplemented with linoleic acid grown in the presence of PHA, compared with those grown in linoleic acid-supplemented medium alone, showed a 40% decrease in palmitic acid and a 55% increase in arachidonic acid in PE. For PC, there were large decreases in stearic acid (40%) and arachidonic acid (57%). Antibody-induced redistribution of surface molecules ('capping') was inhibited by some 14% after incubation with BSA-LA. However, no consistent alterations in PHA-induced cell proliferation were observed. These data suggest that profound alterations of membrane fatty acid composition occur spontaneously during the mitotic cycle, and may be further induced by experimental manipulation, without gross perturbation of cell function.

The etiology of the hypertriglyceridemia in alloxan-diabetic rabbits was studied by two independent methods. Production and removal rates of VLDL triacylglycerol were measured in diabetic rabbits by injection of [3H]palmitate-labelled VLDL. Similarly, triacylglycerol total removal rates were determined in non-diabetic rabbits which were infused with Intralipid to mimic the plasma triacylglycerol concentrations of diabetic rabbits. Compared to nondiabetic rabbits, triacylglycerol removal rats were decreased in diabetic rabbits, particularly at higher levels of plasma triacylglycerol. During cholesterol and triacylglycerol supplementation of the diet, post-heparin plasma lipoprotein lipase activity of diabetic rabbits with severe hypertriglyceridemia averaged 36% of that of nondiabetics, suggesting an impaired triacylglycerol removal capacity. Furthermore, plasma triacylglycerol was inversely related to post-heparin plasma lipoprotein lipase activity among diabetic rabbits. VLDL triacylglycerol production increased with increasing plasma triacylglycerol concentration among diabetic cholesterol-fed rabbits with moderately severe hypertriglyceridemia, but reached an apparent plateau among rabbits with plasma triacylglycerol concentrations from approx. 2000-9000 mg/dl. Thus, severe hypertriglyceridemia in this model of insulin deficiency can be attributed only partially to VLDL hypersecretion, whereas a removal defect, resulting in saturation of the triacylglycerol removal mechanism, appears to be largely responsible. The impaired removal of plasma triacylglycerol is also related to the presence of cholesterol predominantly in lipoproteins of increased size. The data support the hypothesis that protection against atherosclerosis in cholesterol-fed diabetic rabbits results from exclusion of very large cholesterol-containing lipoproteins from the arterial wall.

The kinetics of labeling of lung phosphatidylcholine and disaturated phosphatidylcholine were studied for periods from 0.75--120 min following intravenous injection of radiolabeled palmitic acid and choline into 3-day-old rabbits. The labeled palmitic acid was cleared rapidly from plasma, and rapidly appeared with identical incorporation kinetics in both phosphatidylcholine and disaturated phosphatidylcholine. The 2-acyl positions of both phosphatidylcholine and disaturated phosphatidylcholine were labeled preferentially soon after [14C]palmitic acid injection. The specific activities of palmitic acid in the 2-acyl positions of phosphatidylcholine and disaturated phosphatidylcholine 0.75 min after injection of labeled palmitic acid were 3.4 and 1.9 times, respectively, the specific activities of palmitic acid in the 1-acyl positions. By 120 min the label had randomized between the 1-acyl and 2-acyl positions, and the kinetics of that randomization were defined for both phosphatidylcholine and disaturated phosphatidylcholine. Choline did not pulse label lung phosphatidylcholine or disaturated phosphatidylcholine. The choline label appeared with equal specific activities in both phosphatidylcholine and disaturated phosphatidylcholine. Thus no analysis of the de novo synthesized product via the CDP-choline pathway was possible.

1. Reticulocytosis of 40-50% was obtained in rabbits by daily bleeding. Reticulocytes (plus erythrocytes) were subfractionated into plasma membrane fraction, mitochondria and the post-mitochondrial fraction. 2. In all fractions, fatty acids were incorporated into phospholipids. This process was ATP dependent and represented acylation of lysophospholipids. 3. Incorporation of fatty acids into lysophosphatidic and phosphatidic acids occurred only in the presence of sn-glycerol 3-phosphate and was observed in mitochondria and the post-mitochondrial fraction. It represents a two-step acylation of sn-glycerol 3-phosphate. 4. Incorporation of phosphorylcholine from CDPcholine into phosphatidylcholine was observed in the mitochondrial and the post-mitochondrial fractions. This activity was correlated with NADPH-cytochrome c reductase and was probably connected with the remnants of the endoplasmic reticulum.

Phorbol myristate acetate is known to reproduce the stimulated oxidative activities characteristic of phagocytosis and its initial action is on the cell membrane. In the present study the effect of phorbol myristate acetate on the metabolism of the fatty acyl groups of granulocyte phospholipids was examined and compared with that of phagocytic stimuli. Phorbol myristate acetate stimulated the labeling of phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol by [1-14C]palmitic acid but not by [U-14C]glycerol, whereas starch granules selectively increased the labeling of phosphatidylinositol by both radioactive tracers. Labeled palmitic acid was found at both sn-1- and sn-2-positions of phospholipids and more radioactivity was recovered from the 2-position. The radioactivity at both positions was enhanced in stimulated cells. These data suggest that phorbol myristate acetate increased palmitic acid incorporation into glycerophospholipids by increasing the acylation of the lyso derivatives and that starch granules enhanced the formation of phosphatidylinositol via de novo synthesis and acylation of the lyso derivative as well. Both phorbol myristate acetate and starch granules selectively augmented the incorporation of [1-14C]arachidonic acid into phosphatidylinositol which exhibited the highest specific radioactivity among the phospholipids in control and in stimulated cells. The possible significance of the increased incorporation of arachidonic acid into phosphatidylinositol is discussed.

The incorporation of radiolabeled arachidonic acid and saturated fatty acids into choline-linked phosphoglycerides (PC) of rabbit and human neutrophils was investigated by resolving the individual molecular species by reversed-phase high performance liquid chromatography. PC from neutrophils incubated with a mixture of [3H]arachidonic acid and [14C]stearic or [14C]palmitic acid contains both radiolabels; however, double labeling of individual molecular species is minimal. After labeling for 2 h, the [3H]arachidonate is distributed almost equally between diacyl and 1-O-alkyl-2-acyl species, but it is incorporated into diacyl species containing unlabeled stearate or palmitate at the sn-1 position. In contrast, labeled saturated fatty acids are incorporated only into diacyl species and contain predominantly oleate and linoleate at the sn-2 position. Labeled linoleate is not incorporated into ether-linked species, but is found in the same species as labeled stearate. The findings suggest that mechanisms exist in neutrophils for specific shunting of exogenous arachidonic acid into certain phospholipid molecular species and support the concept that the 1-O-alkyl-2-arachidonoyl species may be a functionally segregated pool of arachidonic acid within the PC of neutrophils.

1. The molecular composition of phosphatidylcholine is similar in the inner and the outer layer of the rat erythrocyte membrane. 2. The rate of exchange of the various molecular classes of phosphatidylcholine between rat plasma and the red cell membrane does not depend on the degree of unsaturation of the different classes. 3. The transposition of the molecular classes of phosphatidylcholine between the inner and the outer layer of the rat erythrocyte membrane is more pronounced for the more unsaturated classes.

When washed human platelets were incubated with [3H]palmitic acid, radioactivity was incorporated into a major 38 kDa doublet and several minor proteins that were resolved on polyacrylamide gels. The radioactivity associated with the proteins remained after extractions with organic solvents, but it was lost after hydroxylamine treatment or mild alkali methanolysis. The products of these reactions were analyzed by thin-layer chromatography and HPLC. They were identified as palmitohydroxamate and methyl palmitate, respectively, indicating that the palmitic acid was covalently linked to the proteins via oxygenester or thioester bonds. In resting platelets, radioactivity was detected in the 38 kDa proteins 2 min after the addition of [3H]palmitic acid. A plateau was reached between 5 and 11 min, at which time radioactivity was also detected in a 23 kDa protein. Thrombin elicited faster and greater incorporation of label into both proteins. Phorbol 12-myristate 13-acetate (PMA) led to a similar, but slower increase of radioactivity in the 38 kDa proteins, while collagen and A23187 were less effective. Enhanced palmitoylation may be closely linked to platelet activation, as suggested by the following observations: (1) in thrombin- or PMA-activated platelets, the time-course of aggregation correlated with the time-course of enhanced palmitoylation of the 38 kDa proteins; (2) in platelets activated by various concentrations of thrombin with or without prostacyclin, aggregation was correlated with the enhanced incorporation of radioactivity into the 38 kDa proteins.

The distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein was measured in vitro by a new method based on the separation of the two proteins by virtue of the binding specificity of concanavalin A for the carbohydrate moiety of alpha-fetoprotein. Human and bovine proteins were investigated. It was found that palmitate and oleate were distributed almost equally between albumin and alpha-fetoprotein, while docosahexaenoate and diethylstilbestrol bound preferentially to alpha-fetoprotein even at an albumin: alpha-fetoprotein ratio of 10:1. The results confirm the binding specificity of alpha-fetoprotein for polyunsaturated fatty acids and also show that alpha-fetoprotein binds diethylstilbestrol much more strongly than albumin does. This suggests that alpha-fetoprotein may play a role in the fetal uptake of diethylstilbestrol.

Non-esterified long-chain fatty acids reduce the extent of hypotonic hemolysis at a certain low concentration range but cause hemolysis at higher concentrations. This biphasic behavior was investigated at different temperatures (0-37 degrees C) for lauric (12:0), myristic (14:0), palmitoleic (16:1), oleic (cis-18:1) and elaidic (trans-18:1) acids. The results are summarized as follows: (A) the fatty acids examined exhibit a high degree of specificity in their thermotropic behavior; (B) oleic acid protects against hypotonic hemolysis even at the highest concentrations, up to 15 degrees C, when it becomes hemolytic, but only in a limited concentration range; (C) elaidic acid does not affect the osmotic stability of erythrocytes up to 20 degrees C, when it starts protecting: above 30 degrees C, it becomes hemolytic at the highest concentrations; (D) palmitoleic acid is an excellent protecting agent at all temperatures in a certain concentration range, becoming hemolytic at higher concentrations; (E) lauric acid protects up to 30 degrees C and becomes hemolytic only above this temperature; (F) myristic acid exhibits an extremely unusual behavior at 30 and 37 degrees C by having alternating concentration ranges of protecting and hemolytic effects; (G) there is a common critical temperature for hemolysis at 30 degrees C for saturated and trans-unsaturated fatty acids; (H) the initial slope of Arrhenius plots of percent hemolysis at the concentration of maximum protection is negative for cis-unsaturated fatty acids and positive for saturated and trans-unsaturated fatty acids.