Arthur A. Spector

Binding of long chain fatty acids to β-lactoglobulin.

Abstractβ-lactoglobulin (BLG), a bovine milk protein that is available commercially in crystalline form, binds long chain free fatty acids (FFA). The binding data were analyzed with a model containing one primary FFA binding site and a large number of weak secondary binding sites. At 37C and pH 7.4, the apparent association constant for binding of FFA to the primary site was of the order of 105 M−1 and that for binding to the secondary sites was approximately 103 M−1. The strength of binding was: palmitate > stearate > oleate > laurate. The affinity of BLG for palmitate increased as the pH of the incubation medium was raised from 6.5 to 8.7 and decreased as the ionic strength of the medium was raised. Palmitate binding was decreased in the presence of 6 M urea and when the protein either was exposed to elevated temperature or was acetylated prior to incubation. BLG took up methyl palmitate, cetyl alcohol, hexadecane and cholesterol to a lesser extent than FFA. Binding of FFA to BLG was associated with a small increase in the intensity of the fluorescent emission of the protein at 333 mμ. BLG can serve as an FFA acceptor or carrier in biological experiments. FFA released from adipose tissue during in vitro incubation was taken up by BLG. Net transfer of fatty acid to the incubation medium ceased when the molar ratio of FFA to BLG exceeded 1.1.14C-1-Palmitate bound to BLG was taken up by Ehrlich ascites tumor cells in vitro. At a given palmitate-protein molar ratio, much more labeled fatty acid was taken up by these cells from media containing BLG than from those containing bovine albumin, apparently because FFA is bound less firmly to BLG than to albumin.

Workshop on the impact of dietary cholesterol on plasma lipoproteins and atherogenesis.

Aworkshop entitled The Impact of Dietary Cholesterol on Plasma Lipoproteins and Atherogenesis was held in Bethesda, Maryland on July 1-3, 1986. This workshop was cosponsored by the National Heart, Lung, and Blood Institute and the Agricultural Research Service, United States Department of Agriculture (USDA). Its purpose was to review existing data relating dietary cholesterol to coronary heart disease (CHD) and to define needs for future research on this issue. The topics discussed ranged from basic science to epidemiology and included investigations using both humans and laboratory animals. A possible relation between dietary cholesterol and atherosclerosis was first brought to light in laboratory animals. The feeding of cholesterol can induce marked hypercholesterolemia and atherosclerosis in many species. This observation and the susceptibility of nonhuman primates to the plasma cholesterol-raising effects of dietary cholesterol has influenced thinking about the role of dietary cholesterol in the genesis of CHD in humans. For many years, however, skeptics have questioned whether findings in laboratory animals can be extended to humans. Clinical investigations have revealed that high intakes of cholesterol in humans do not induce a marked hypercholesterolemia. Thus, some workers have suggested that humans are basically resistant to dietary cholesterol, and they contend that CHD risk is not reduced by restricting dietary cholesterol. This workshop examined the bases for these opposing views by considering the known or potential effects of dietary cholesterol.

Phosphatidylserine in the Brain: Metabolism and Function

Phosphatidylserine (PS) is the major anionic phospholipid class particularly enriched in the inner leaflet of the plasma membrane in neural tissues. PS is synthesized from phosphatidylcholine or phosphatidylethanolamine by exchanging the base head group with serine, and this reaction is catalyzed by phosphatidylserine synthase 1 and phosphatidylserine synthase 2 located in the endoplasmic reticulum. Activation of Akt, Raf-1 and protein kinase C signaling, which supports neuronal survival and differentiation, requires interaction of these proteins with PS localized in the cytoplasmic leaflet of the plasma membrane. Furthermore, neurotransmitter release by exocytosis and a number of synaptic receptors and proteins are modulated by PS present in the neuronal membranes. Brain is highly enriched with docosahexaenoic acid (DHA), and brain PS has a high DHA content. By promoting PS synthesis, DHA can uniquely expand the PS pool in neuronal membranes and thereby influence PS-dependent signaling and protein function. Ethanol decreases DHA-promoted PS synthesis and accumulation in neurons, which may contribute to the deleterious effects of ethanol intake. Improvement of some memory functions has been observed in cognitively impaired subjects as a result of PS supplementation, but the mechanism is unclear.

Synaptamide, endocannabinoid-like derivative of docosahexaenoic acid with cannabinoid-independent function.

Docosahexaenoylethanolamide, the structural analog of the endogenous cannabinoid receptor ligand anandamide, is synthesized from docosahexaenoic acid (DHA) in the brain. Although docosahexaenoylethanolamide binds weakly to cannabinoid receptors, it stimulates neurite growth, synaptogenesis and glutamatergic synaptic activity in developing hippocampal neurons at concentrations of 10-100 nM. We have previously proposed the term synaptamide for docosahexaenoylethanolamide to emphasize its potent synaptogenic activity and structural similarity to anandamide. Synaptamide is subjected to hydrolysis by fatty acid amide hydrolase, and can be oxygenated to bioactive metabolites. The brain synaptamide content is dependent on the dietary DHA intake, suggesting an endogenous mechanism whereby diets containing adequate amounts of omega-3 fatty acids improve synaptogenesis in addition to well-recognized anti-inflammatory effects.

A procedure for computer analysis of data from macromolecule-ligand binding studies

Abstract A procedure for fitting macromolecule-ligand binding data to a mathematical model using computer techniques is described. The computer algorithm for carrying out this procedure together with a flow diagram of the main program are presented. Using this procedure, the choice of an appropriate model is made from computed results, and the subjective bias often associated with hand-drawn graphical methods is avoided. In addition, estimates of the statistical standard errors of the binding constants are obtained, and information concerning the validity of the model is provided. The output obtained from the computer is displayed in both tabular and graphical form.

Effects of free fatty acid on the fluorescence of bovine serum albumin

Abstract The ultraviolet fluorescence emission spectrum of bovine serum albumin was altered when free fatty acid (FFA) was added to the protein. This occurred when FFA was taken up from aqueous soap solutions, n -heptane solutions, rat epididymal fat pads, or fat emulsions undergoing enzymatic hydrolysis. As the molar ratio of FFA to albumin increased, the maximum emission was shifted to a shorter wave length and the fluorescence intensity was decreased. The blue shift did not exceed 7 mμ, and the maximum reduction in fluorescence intensity was less than 50% even when the molar ratio of FFA to albumin was high. Qualitatively similar results were obtained with FFA of 6–22 C atoms, but smaller effects occurred when FFA of less than 10 C atoms were used. FFA produced little or no change in the fluorescence intensity when the pH was less than 5.5 or greater than 10. FFA produced changes in the fluorescence spectrum of porcine albumin but had little effect on that of human, rabbit, equine or canine albumins.

Discovery of essential fatty acids

Dietary fat was recognized as a good source of energy and fat-soluble vitamins by the first part of the 20th century, but fatty acids were not considered to be essential nutrients because they could be synthesized from dietary carbohydrate. This well-established view was challenged in 1929 by George and Mildred Burr who reported that dietary fatty acid was required to prevent a deficiency disease that occurred in rats fed a fat-free diet. They concluded that fatty acids were essential nutrients and showed that linoleic acid prevented the disease and is an essential fatty acid. The Burrs surmised that other unsaturated fatty acids were essential and subsequently demonstrated that linolenic acid, the omega-3 fatty acid analog of linoleic acid, is also an essential fatty acid. The discovery of essential fatty acids was a paradigm-changing finding, and it is now considered to be one of the landmark discoveries in lipid research.

Effect of carnitine on free fatty acid utilization in ehrlich ascites tumor cells

Abstract The oxidation of palmitate-1- 14 C to 14 CO 2 in homogenates of Ehrlich ascites tumor cells was increased approximately 2.5 times by the addition of carnitine. Carnitine palmityltransferase and long-chain fatty acid thiokinase activities were demonstrated in this homogenate. It is postulated that, as in non-enoplastic tissues, an acylcarnitine intermediate probably is involved in the oxidation of long-chain fatty acids by the tumor homogenate. Radioactive carnitine was taken up by the intact tumor cell, and a small fraction of this was incorporated into long-chain acylcarnitine. When exogenous palmitate was available, the incorporation of radioactive carnitine into long-chain acylcarnitine was doubled. However, exogenous palmitate-1- 14 C oxidation by the intact tumor cell was not stimulated by addition of carnitine to the incubation medium, and deoxycarnitine did not inhibit palmitate oxidation by the intact cell. Added carnitine also had no effect on palmitate-1- 14 C incorporation into the total lipid, phospholipid, triglyceride, or free fatty acid fractions of the intact cell.

N-Docosahexaenoylethanolamine: A neurotrophic and neuroprotective metabolite of docosahexaenoic acid

N-Docosahexaenoylethanolamine (synaptamide) is an endocannabinoid-like metabolite endogenously synthesized from docosahexaenoic acid (DHA, 22:6n-3), the major omega-3 polyunsaturated fatty acid present in the brain. Although its biosynthetic mechanism has yet to be established, there is a closely linked relationship between the levels of synaptamide and its precursor DHA in the brain. Synaptamide at nanomolar concentrations promotes neurogenesis, neurite outgrowth and synaptogenesis in developing neurons. Synaptamide also attenuates the lipopolysaccharide-induced neuroinflammatory response and reduces the deleterious effects of ethanol on neurogenic differentiation of neural stem cells (NSCs). These actions are mediated by a specific target receptor of synaptamide GPR110 (ADGRF1), a G-protein coupled receptor that is highly expressed in NSCs and the brain during development. Synaptamide binding to GPR110 induces cAMP production and phosphorylation of protein kinase A (PKA) and the cAMP response element binding protein (CREB). This signaling pathway leads to the expression of neurogenic and synaptogenic genes and suppresses the expression of proinflammatory genes. The GPR110-dependent cellular effects of synaptamide are recapitulated in animal models, suggesting that synaptamide-derived mechanisms may have translational implications. The synaptamide bioactivity transmitted by newly deorphanized GPR110 provides a novel target for neurodevelopmental and neuroprotective control as well as new insight into mechanisms for DHAs beneficial effects on the central nervous system.