Fatty acid enhancement of human serum albumin binding properties. A spin label study.

The introduction of a new spin-labeled anionic ligand, 1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene, is reported. Under the experimental conditions, the first molar equivalent of this ligand is 93% bound to human serum albumin. With the addition of palmitate, the free spin label concentration decreases greatly, by almost 80%, in the presence of a fatty acid:albumin ratio of 3:1 to 4:1. The spectral characteristics of the bound spin label are also affected. The changes seen in the intensity of and the splitting between the high and low field extrema are indicative of perturbations of the protein molecule. It is seen then that the binding of each molar equivalent of fatty acid effects the conformation state of albumin and allosterically affects albumin binding properties. Computer spectral subtractions, furthermore, suggest that the binding of the first molar equivalent of palmitate specifically increases the affinity of the first two 1-gamma-amino-butyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene binding sites. The present results indicate that fluctuations in serum free fatty acid levels within the physiological range may have a major modulatory effect on the free serum levels of certain drugs and/or physiological substances that bind to albumin.     

High affinity of alpha-foetoprotein for arachidonate and other fatty acids.

Fatty acid analysis of purified bovine alpha-foetoprotein showed it to contain 2.7 mol of fatty acid/mol of alpha-foetoprotein. Purified alpha-foetoprotein focused at isoelectric point 4.8. Removal of bound ligands from alpha-foetoprotein by charcoal treatment changed its isoelectric point to 5.2. This change could be reversed by addition of exogenous fatty acids to the defatted alpha-foetoprotein. Albumin isolated from the same foetal calf serum source as alpha-foetoprotein contained 1.4 mol of fatty acid/mol of protein. alpha-Foetoprotein and albumin contained comparable amounts of fatty acids with 14 to 18 carbon atoms, but alpha-foetoprotein contained 16 times as much of the long-chain polyunsaturated fatty acids as albumin. alpha-Foetoprotein was found to have slightly higher affinity for palmitate and linoleate and severalfold higher affinity for arachidonate than albumin. These findings suggest that alpha-foetoprotein may play a role in the foetal metabolism of the long-chain polyunsaturated fatty acids.     

Expression of rat intestinal fatty acid-binding protein in Escherichia coli. Purification and comparison of ligand binding characteristics with that of Escherichia coli-derived rat liver fatty acid-binding protein

Rat intestinal fatty acid-binding protein (I-FABP) is an abundant, 15,124-Da polypeptide found in the cytosol of small intestinal epithelial cells (enterocytes). It is homologous to rat liver fatty acid-binding protein (L-FABP), a 14,273-Da cytosolic protein which is found in enterocytes as well as hepatocytes. It is unclear why the small intestinal epithelium contains two abundant fatty acid-binding proteins. A systematic comparative analysis of the ligand binding characteristics of the two FABPs has not been reported. To undertake such a study we expressed the coding region of a full length I-FABP cDNA in Escherichia coli and purified large quantities of the protein. We also purified rat L-FABP from a similar, previously described expression system (Lowe, J. B., Strauss, A. W., and Gordon, J. I. (1984) J. Biol. Chem. 259, 12696-12704). Analysis of fatty acids associated with each of the homogeneous E. coli-derived FABPs suggested that the two proteins differed in their ligand binding specificity and capacity. All of the fatty acids associated with I-FABP were saturated while 30% of the E. coli fatty acids bound to L-FABP were unsaturated (16:1, 18:1, 18:2). We directly analyzed the ability of I- and L-FABP to bind fatty acids of different chain length and degree of saturation using a hydroxyalkoxypropyl dextran-based assay. Scatchard analysis revealed that each mole of L-FABP can bind up to 2 mol of long chain fatty acid while each mole of I-FABP can bind only 1 mole of fatty acid. L-FABP exhibited a relatively higher affinity for unsaturated fatty acids (oleate, arachidonate) than for saturated fatty acid (palmitate). By contrast, we were not able to detect a significant difference in the affinity of I-FABP for palmitate, oleate, and arachidonate. Neither protein exhibited any appreciable affinity for fatty acids whose chain length was less than C16. The observed differences in ligand affinities and capacities suggest that these proteins may have distinct roles in metabolism and/or compartmentalization of fatty acids within enterocytes.     

The effect of endogenous essential and nonessential fatty acids on the uptake and subsequent agonist-induced release of arachidonate

We have demonstrated that the uptake and agonist-induced release of a pulse of arachidonate are influenced by the size and composition of preexisting endogenous fatty acid pools. EFD-1 cells, an essential fatty acid-deficient mouse fibrosarcoma cell line, were incubated with radiolabeled (14C or 3H] arachidonate, linoleate, eicosapentaenoate (EPA), palmitate, or oleate in concentrations of 0-33 microM for 24 h. After 24 h, the cells were pulsed with 0.67 microM radiolabeled (3H or 14C, opposite first label) arachidonate for 15 min and then stimulated with 10 microM bradykinin for 4 min. Because EFD-1 cells contain no endogenous essential fatty acids, we were able to create essential fatty acid-repleted cells for which the specific activity of the newly constructed endogenous essential fatty acid pool was known. Loading the endogenous pool with the essential fatty acids arachidonate, eicosapentaenoate, or linoleate (15-20 nmol of fatty acid incorporated/10(6) cells) decreased the uptake of a pulse of arachidonate from 200 to 100 pmol/10(6) cells but had no effect on palmitate uptake. The percent of arachidonate incorporated during the pulse which was released upon agonist stimulation increased 2-fold (4-8%) as the endogenous pool of essential fatty acids was increased from 0 to 15-20 nmol/10(6) cells. This 8% release was at least 3-fold greater than the percent release from the various endogenous essential fatty acid pools. In contrast, loading the endogenous pool with the nonessential fatty acids oleate or palmitate to more than 2-3 times their preexisting cellular level had no effect on the uptake of an arachidonate pulse. Like the essential fatty acids, increasing endogenous oleate increased (by 2-fold) the percent release of arachidonate incorporated during the pulse, whereas endogenous palmitate had no effect on subsequent agonist-induced release from this arachidonate pool. These studies show that preexisting pools of essential and nonessential fatty acids exert different effects on the uptake and subsequent releasability of a pulse of arachidonate.     

Phospholipid metabolism of stimulated lymphocytes. Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [3H]arachidonate and [14C]oleate or [3H]arachidonate and [14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3-7-fold) and phosphatidylethanolamine (2-3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Mouse alpha 1-fetoprotein and albumin. A comparison of their binding properties with estrogen and fatty acid ligands

The binding of estradiol-17 beta (E2), diethylstilbestrol (DES), and polyene fatty acids, in particular arachidonate (C20:4), to alpha 1-fetoprotein (alpha-FP) and albumin purified from mouse embryo sera was studied using equilibrium dialysis and electrophoretic techniques. E2, arachidonate, and DES all bind to alpha-FP, but with decreasing strength. E2 is a high affinity, low capacity ligand (Ka approximately 0.8 X 10(8) M-1 and approximately 0.3 sites/mol of alpha-FP at 25 degrees C); arachidonate is a weaker ligand disposing of more sites (Ka approximately 0.3 X 10(7) M-1 and 4-5 sites/mol of alpha-FP); the binding of DES is of comparatively low affinity and capacity (Ka approximately 0.2 X 10(7) M-1 and n approximately 0.7/mol of alpha-FP). In spite of different structures and equilibrium parameters, E2, DES, and arachidonate are able to compete with each other for binding to the fetoprotein. The C22:4 and C22:6 fatty acids are also efficient concentration-dependent inhibitors of E2 or DES binding. Albumin binds the fatty acids and DES, but equilibrium parameters are different from those of alpha-FP. In particular, arachidonate is a better ligand for albumin, where it interacts with at least two classes of apparent sites (Ka1 approximately 0.3 X 10(8) M-1 and n1 approximately 1; Ka2 approximately 0.2 X 10(7) M-1 and n2 approximately 30). In contrast to alpha-FP, albumin virtually does not bind E2. Also, no competition could be demonstrated between DES and fatty acid ligands for binding to albumin. None of the studied interactions, with either albumin or alpha-FP, was modified even by high doses of bilirubin. The possible functions of the various binding activities present in fetal sera in the process of growth are discussed.     

Disproportionately higher levels of myocardial docosahexaenoate and elevated levels of plasma and liver arachidonate in hyperthyroid rats

The effects of hyperthyroidism on the metabolism and distribution of polyunsaturated fatty acids in rats were investigated. The animals were fed diets containing an equal amount (1% each) of linoleate and linolenate. Although the hepatic and plasma levels of the linolenate family of acids were not greatly affected by the hyperthyroidism, the heart of the hyperthyroid rat contained 425% more docosahexaenoate than did that of its euthyroid control. The hyperthyroidism was accompanied by accumulations of 85, 105, and 114% more arachidonic acid in the heart, plasma, and liver, respectively. Nevertheless, most of the total increases in plasma and liver fatty acids were due to the greater accumulations of palmitic, stearic, and oleic acids; the hepatic level of oleate was elevated by 204%. Hyperthyroid rats had 106% more total fatty acids in their hearts, this increase being due largely to the greater accumulation of polyunsaturated acids. The thyroid hormone appears to accelerate the biosynthesis of both arachidonate and docosahexaenoate, and these endogenous polyunsaturated acids are then selectively incorporated into the cardiovascular tissues. Other possible relationships between thyroid action and tissue polyenoic acids in "cold-stressed" animals are discussed.     

Phospholipid metabolism of stimulated lymphocytes: Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [³H]arachidonate and [¹⁴C]oleate or [³H]arachidonate and [¹⁴C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3–7-fold) and phosphatidylethanolamine (2–3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Lipids and lipid metabolism in the brown alga, Fucus serratus

The lipids of the brown alga Fucus serratus were isolated, identified and quantified. The major acyl lipids were the three glycosylglycerides, diacylgalactosylglycerol, diacyldigalactosylglycerol and diacylsulphoquinovosylglycerol. These represent over 70% of the total acyl lipids. The fatty acid compositions of the major lipids were examined and most showed rather distinctive fatty acid contents. For example, diacylgalactosylglycerol was enriched in n-3 polyunsaturated fatty acids while phosphatidylcholine and phosphatidylethanolamine had very high levels of arachidonate. Phosphatidylglycerol contained the unusual trans-Δ3-hexadecenoic acid. The labelling of lipids and fatty acids from [¹⁴C]acetate was examined and the distribution of label between individual components as a function of the incubation period and in algae collected at different times of the year is reported. Algae collected in the winter incorporated much more radioactivity into non-esterified fatty acids when compared to algae collected in the summer. All algae could label myristate, palmitate, stearate and oleate at high rates. Longer incubation times allowed the labelling of polyunsaturated fatty acids such as linoleic acid.     

Problems with essential fatty acids: time for a new paradigm?

The term ‘essential fatty acid’ is ambiguous and inappropriately inclusive or exclusive of many polyunsaturated fatty acids. When applied most rigidly to linoleate and -linolenate, this term excludes the now well accepted but conditional dietary need for two long chain polyunsaturates (arachidonate and docosahexaenoate) during infancy. In addition, because of the concomitant absence of dietary -linolenate, essential fatty acid deficiency is a seriously flawed model that has probably led to significantly overestimating linoleate requirements. Linoleate and -linolenate are more rapidly β-oxidized and less easily replaced in tissue lipids than the common ‘non-essential’ fatty acids (palmitate, stearate, oleate). Carbon from linoleate and -linolenate is recycled into palmitate and cholesterol in amounts frequently exceeding that used to make long chain polyunsaturates. These observations represent several problems with the concept of ‘essential fatty acid’, a term that connotes a more protected and important fatty acid than those which can be made endogenously. The metabolism of essential and non-essential fatty acids is clearly much more interconnected than previously understood. Replacing the term ‘essential fatty acid’ by existing but less biased terminology, i.e. polyunsaturates, ω3 or ω6 polyunsaturates, or naming the individual fatty acid(s) in question, would improve clarity and would potentially promote broader exploration of the functional and health attributes of polyunsaturated fatty acids.     

Ligand-binding properties of albumin Parklands: Asp365----His

An albumin variant, isolated from the plasma of a patient with bisalbuminemia, was compared with albumin A from the same patient for binding of long-chain fatty acids and bilirubin. No differences in binding of [14C]palmitate, cis-parinaric acid or bilirubin could be detected for the variant form. These results suggest that the region adjacent to residue 365 is unlikely to be part of a major binding site for any of these ligands.     

Metabolism of polyunsaturated fatty acids and ketogenesis: an emerging connection

This paper summarizes the emerging literature indicating that at least two polyunsaturated fatty acids (PUFA; linoleate, alpha-linolenate) are moderately ketogenic and that via ketone bodies significant amounts of carbon are recycled from these fatty acids into de novo synthesis of lipids including cholesterol, palmitate, stearate and oleate. This pathway (PUFA carbon recycling) is particularly active in several tissues during the suckling period when, depending on the tissue, >200 fold more carbon from alpha-linolenate can be recycled into newly synthesized lipids than is used to make docosahexaenoate. At least in rats, PUFA carbon recycling also occurs in adults and even during extreme linoleate deficiency. Hence, this pathway should be considered an obligatory component of PUFA metabolism. It is still speculative but part of the clinical benefit of the very high fat ketogenic diet in intractable seizures may be achieved by raising plasma levels of PUFA that have anti-seizure effects, especially arachidonate and docosahexaenoate. Hence, in addition to some PUFA being ketogenic substrates, the state of ketosis involves potentially beneficial changes in PUFA homeostasis. Both the molecular controls on these pathways and their clinical significance still need elucidation.     

Decreased content of docosahexaenoate and arachidonate in plasma phospholipids in Usher's syndrome

Docosahexaenoate and arachidonate were found to be significantly decreased in plasma phospholipids from Usher's syndrome patients. The fatty acid content of plasma triacylglycerols was not changed in these patients. Usher's syndrome, an autosomal recessive disorder, involves an inherited visual cell degeneration. Photoreceptor membranes are richly endowed with docosahexaenoate and arachidonate, and a metabolic defect affecting these polyunsaturated fatty acids may occur. Moreover, blindness may be due, at least partially, to an alteration in the unsaturated phospholipids of photoreceptor membranes.     

Human alpha-fetoprotein-fatty acid interaction

Human alpha-fetoprotein (AFP) is able to bind arachidonic acid with high affinity Ka = 10(7)M-1 and a limited number of binding sites NS = 3. Thirty different fatty acids were tested by competition using labelled arachidonic acid as tracer. This experiment demonstrated a great specificity for the fatty acid-AFP interaction. Only polyunsaturated long chain fatty acids were able to bind to AFP with high affinity. The metabolic products of arachidonic acid i.e. prostaglandins presented no affinity for the AFP molecule.     

Determination of the distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein by concanavalin A affinity chromatography

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.     

Steroid modulation of human serum albumin binding properties. A spin label study.

The binding isotherm and unique electron spin resonance spectral characteristics of a monoanionic spin label (1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) and a dianionic spin label (1-glutamate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) are used to prove the steroid modulation of serum albumin binding properties. Effects of a selected number of steroids (progesterone, testosterone, estradiol, aldosterone, estriol, corticosterone, deoxycorticosterone, hydrocortisone, and cortisone) on the binding isotherm of the monoanionic spin label binding to serum albumin have been determined. At the steroid/albumin ratio of 0.5 to 1, progesterone, testosterone, and estradiol enhance binding of the spin label at all concentrations studied. However, the remaining steroids exert an inhibitory effect at low spin label/albumin ratios and an enhancement effect at high spin label/albumin ratios. Progesterone and cortisone effects on the resonance spectra of the spin label bound to serum albumin confirm the enhancement and displacement properties of these ligands. Thus, like fatty acids, steroids may bind to either the primary or secondary bilirubin binding sites and also allosterically perturb the binding properties of serum albumin. The in vivo importance of the steroid-albumin interaction is discussed.     

Brain lipid metabolism in the cPLA2 knockout mouse

We examined brain phospholipid metabolism in mice in which the cytosolic phospholipase A(2) (cPLA(2,) Type IV, 85 kDa) was knocked out (cPLA(2)(-/-) mice). Compared with controls, these mice demonstrated altered brain concentrations of several phospholipids, reduced esterified linoleate, arachidonate, and docosahexaenoate in choline glycerophospholipid, and reduced esterified arachidonate in phosphatidylinositol. Unanesthetized cPLA(2)(-/-) mice had reduced rates of incorporation of unlabeled arachidonate from plasma and from the brain arachidonoyl-CoA pool into ethanolamine glycerophospholipid and choline glycerophospholipid, but elevated rates into phosphatidylinositol. These differences corresponded to altered turnover and metabolic loss of esterified brain arachidonate. These results suggests that cPLA(2) is necessary to maintain normal brain concentrations of phospholipids and of their esterified polyunsaturated fatty acids. Reduced esterified arachidonate and docosahexaenoate may account for the resistance of the cPLA(2)(-/-) mouse to middle cerebral artery occlusion, and should influence membrane fluidity, neuroinflammation, signal transduction, and other brain processes.     

Location of long chain fatty acid-binding sites of bovine serum albumin by affinity labeling

Affinity labeling with palmitic acid was used to identify long chain fatty acid-binding sites of bovine serum albumin. [1-14C]Palmitic acid was activated by esterification with N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's Reagent K). The product was purified by chromatography and shown to compete with unesterified fatty acids for binding sites on bovine serum albumin. Activated [14C]palmitic acid coupled covalently to albumin producing [14C]palmitoyl-albumins containing from 0.12 to a maximum of 6.9 mol of attached label per mol of albumin. The presence of the covalently attached affinity label depressed binding of other long chain fatty acids to albumin. Albumin carrying 1 eq. of [14C]palmitate was cleaved using cyanogen bromide, pepsin, and trypsin. Radioactive peptides were isolated by high pressure liquid chromatography. Three peptides accounted for greater than 90% of the label. Residues labeled with [14C]palmitate were identified as Lys-116, Lys-349 and Lys-473, and the relative distribution of label was 10, 45, and 45% respectively, consistent with the presence of two strong binding sites in the COOH-terminal half of albumin and a somewhat weaker site in the NH2-terminal half.