Omega-3 fatty acids and neuropsychiatric disorders

Epidemiological evidence suggests that dietary consumption of the long chain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), commonly found in fish or fish oil, may modify the risk for certain neuropsychiatric disorders. As evidence, decreased blood levels of omega-3 fatty acids have been associated with several neuropsychiatric conditions, including Attention Deficit (Hyperactivity) Disorder, Alzheimer's Disease, Schizophrenia and Depression. Supplementation studies, using individual or combination omega-3 fatty acids, suggest the possibility for decreased symptoms associated with some of these conditions. Thus far, however, the benefits of supplementation, in terms of decreasing disease risk and/or aiding in symptom management, are not clear and more research is needed. The reasons for blood fatty acid alterations in these disorders are not known, nor are the potential mechanisms by which omega-3 fatty acids may function in normal neuronal activity and neuropsychiatric disease prevention and/or treatment. It is clear, however, that DHA is the predominant n-3 fatty acid found in the brain and that EPA plays an important role as an anti-inflammatory precursor. Both DHA and EPA can be linked with many aspects of neural function, including neurotransmission, membrane fluidity, ion channel and enzyme regulation and gene expression. This review summarizes the knowledge in terms of dietary omega-3 fatty acid intake and metabolism, as well as evidence pointing to potential mechanisms of omega-3 fatty acids in normal brain functioning, development of neuropsychiatric disorders and efficacy of omega-3 fatty acid supplementation in terms of symptom management.     

A method for quantitative acylcarnitine profiling in human skin fibroblasts using unlabelled palmitic acid: diagnosis of fatty acid oxidation disorders and differentiation between biochemical phenotypes of MCAD deficiency

Inherited disorders of fatty acid oxidation are a group of acute life-threatening but treatable disorders, clinically complicated by severe hypoketotic hypoglycemia precipitated by prolonged fasting. Among them, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is by far the most frequent disorder. Here we report a modified method for quantitative acylcarnitine profiling by electrospray ionisation-tandem mass spectrometry (ESI-MS-MS) in human skin fibroblasts using unlabelled palmitic acid as substrate. The reliability of this method was tested in cultured skin fibroblasts from previously diagnosed patients with specific carnitine cycle and fatty acid beta-oxidation defects. Furthermore, acylcarnitine profiling was investigated in fibroblasts and dried blood spots from patients with different variants of MCAD deficiency. ESI-MS-MS-based investigation of cultured skin fibroblasts from patients with disorders of fatty acid oxidation revealed a pathognomonic acylcarnitine profiling. In addition, this method delineated different variants of MCAD deficiency, i.e. mild and classical. The octanoylcarnitine (C8)-to-decanoylcarnitine (C10) and C8-to-acetylcarnitine (C2) ratios were the most specific markers to differentiate mild and classical forms of MCAD deficiency in fibroblasts. Similar results were obtained by quantitative acylcarnitine profiling in dried blood spots. In conclusion, this novel technique is a powerful tool for the investigation of fatty acid oxidation disorders under standardized conditions in fibroblasts.     

Identification of ester-linked fatty acids of bacterial endotoxins by negative ion fast atom bombardment mass spectrometry

Negative ion fast atom bombardment mass spectrometry (NI-FAB/MS) was employed to characterize the fatty acids esterified to the lipid A backbone of lipopolysaccharides (LPS) of gram-negative bacteria. LPS and their chemically derived lipid A produced readily detectable fragment ions characteristic of fatty acids. The NI-FAB/MS method is specific, yielding ions indicative of ester- but not of amide-bound fatty acids. The mass spectra of Enterobacteriaceae LPS revealed the presence of lauric (m/z 199), myristic (m/z 227), palmitic (m/z 255), and 3-hydroxymyristic (m/z 243) acids. Pseudomonas aeruginosa LPS gave distinctive fragment ions indicative of 3-hydroxydecanoic (m/z 187), lauric, and 2-hydroxylauric (m/z 215) acids. The Neisseria gonorrhoeae LPS could be distinguished from the others due to the presence of ester-linked 3-hydroxylauric acid. All of the LPS gave abundant ions of m/z 177 and 159, which were derived from diphosphoryl substituents. The use of NI-FAB/MS thus allowed rapid identification of lipid A esterified fatty acids without chemical derivatization or gas chromatographic analysis.     

Changes in cerebral membrane lipid composition and fluidity during thioacetamide-induced hepatic encephalopathy

Lipids are an essential structural and functional component of cellular membranes. Changes in membrane lipid composition are known to affect the activities of many membrane-associated enzymes, endocytosis, exocytosis, membrane fusion and neurotransmitter uptake, and have been implicated in the pathophysiology of many neurodegenerative disorders. In the present study, we investigated changes in the lipid composition of membranes isolated from the cerebral cortex of rats treated with thioacetamide (TAA), a hepatotoxin that induces fulminant hepatic failure (FHF) and thereon hepatic encephalopathy (HE). HE refers to acute neuropsychiatric changes accompanying FHF. The estimation of membrane phospholipids, cholesterol and fatty acid content in cerebral cortex membranes from TAA-treated rats revealed a decrease in cholesterol, phosphatidylserine, sphingomyelin, a monounsaturated fatty acid, namely oleic acid, and the polyunsaturated fatty acids gamma-linolenic acid, decosa hexanoic acid and arachidonic acid compared with controls. Assessment of membrane fluidity with pyrene, 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene revealed a decrease in the annular membrane fluidity, whereas the global fluidity was unaffected. The level of the thiobarbituric acid reactive species marker for lipid peroxidation also increased in membranes from TAA-treated rats, thereby indicating the prevalence of oxidative stress. Results from the present study demonstrate gross alterations in cerebral cortical membrane lipid composition and fluidity during TAA-induced HE, and their possible implications in the pathogenesis of this condition are also discussed.     

Pyruvylated glycolipids from Mycobacterium smegmatis. Nature and location of the lipid components

The dipyruvylated glycolipid from Mycobacterium smegmatis (Saadat, S., and Ballou, C.E. (1983) J. Biol. Chem. 258, 1813-1818) has been shown to have the following structure in which FA1 is tetra- or hexadecanoic acid and FA2 is 2,4-dimethyl-2-eicosenoic acid. (formula; see text) The fast atom bombardment mass spectrum showed two major ions [M - H]- at m/z 1511 and 1539 (Mr 1512 and 1540) in a ratio of 1.4:1, suggesting that the glycolipid was a mixture of homologs that differed in fatty acid composition by 2 methylene groups. Analysis revealed C14, C16, and C22 fatty acids in ratios of 0.6:0.4:1.0, indicating that 60% of the molecules contained a C14 and C22 fatty acid whereas 40% contained a C16 and C22 fatty acid. The fragmentation pattern showed that a single glucose unit along with the smaller fatty acid could be lost to yield a tetrasaccharide with attached C22 fatty acid, and a second fragmentation yielded a trisaccharide containing 2 pyruvic acids but without attached fatty acid. The C14 and C16 fatty acids were identified as myristic and palmitic acid, whereas the C22 fatty acid was 2,4-dimethyl-2-eicosenoic acid. Precise localization of the fatty acids came from periodate oxidation and methylation analysis.     

Clinical applications of tandem mass spectrometry: ten years of diagnosis and screening for inherited metabolic diseases

This paper reviews the clinical applications of tandem mass spectrometry (MS–MS) in diagnosis and screening for inherited metabolic diseases in the last 10 years. The broad-spectrum of diseases covered, specificity, ease of sample preparation, and high throughput provided by the MS–MS technology has led to the development of multi-disorder newborn screening programs in many countries for amino acid disorders, organic acidemias, and fatty acid oxidation defects. Issues related to sample acquisition, sample preparation, quantification of metabolites, and validation are discussed. Our current experience with the technique in screening is presented. The application of MS–MS in selective screening has revolutionized the field and made a major impact on the detection of certain disease classes such as the fatty acid oxidation defects. New specific and rapid MS–MS and LC–MS–MS methods for highly polar small molecules are supplementing or replacing some of the classical GC–MS methods for a multitude of metabolites and disorders. New exciting applications are appearing in fields of prenatal, postnatal, and even postmortem diagnosis. Examples for pitfalls in the technique are also presented.     

Mechanisms for ion formation during the electron impact-mass spectrometry of picolinyl ester and 4,4-dimethyloxazoline derivatives of fatty acids

Mass spectral studies have been conducted with isotopically stable labelled and fluorinated picolinyl esters and 4,4-dimethyloxazoline (DMOX) derivatives of fatty acids in order to establish mechanisms of ion formation. Reciprocal hydrogen transfer is shown to be involved in the formation of the ion at m/z 126 with dimethyloxazoline derivatives and for the ion at m/z 164 with picolinyl esters. Inclusion of a fluorine atom alpha to the carboxyl of a fatty acid has been demonstrated to enhance rearrangements for expulsion of internal chain fragments with both methyl ester and dimethyloxazoline derivatives. When two fluorine atoms are inserted into the alpha position a similar rearrangement has been shown to occur with picolinyl esters, although not nearly to the same extent as that observed with either of the other derivatives. Mechanisms for such rearrangements are proposed and discussed. With fatty acid dimethyloxazoline derivatives the M-15 ion arises solely from the loss of a methyl radical from the ring and the M-43 ion has at least three different mechanisms of formation. Such rearrangements make it difficult to establish the identity of the terminal moiety of the alkyl chain. In mass spectrometry terms the picolinyl ester would seem to be the superior derivative for structural characterisation of fatty acids.     

Fatty acids and sterols from Cymbopogon martinii var. motia roots

The hexane and methanol extracts of the roots of Cymbopogon martinii var. motia have been investigated to afford mainly fatty acids and common sterols. A new hydroxy unsaturated fatty acid, namely, 16-hydroxypentacos-14(z)-enoic acid, has also been isolated.     

A Common STEP in the Synaptic Pathology of Diverse Neuropsychiatric Disorders

Synaptic function is critical for proper cognition, and synaptopathologies have been implicated in diverse neuropsychiatric disorders. STriatal-Enriched protein tyrosine Phosphatase (STEP) is a brain-enriched tyrosine phosphatase that normally opposes synaptic strengthening by dephosphorylating key neuronal signaling molecules. STEP targets include N-methyl D-aspartate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), as well as extracellular signal-regulated kinase (ERK) and the tyrosine kinase Fyn. STEP-mediated dephosphorylation promotes the internalization of NMDARs and AMPARs and the inactivation of ERK and Fyn.Regulation of STEP is complex, and recent work has implicated STEP dysregulation in the pathophysiology of several neuropsychiatric disorders. Both high levels and low levels of STEP are found in a diverse group of illnesses. This review focuses on the role of STEP in three disorders in which STEP levels are elevated: Alzheimer’s disease, fragile X syndrome, and schizophrenia. The presence of elevated STEP in all three of these disorders raises the intriguing possibility that cognitive deficits resulting from diverse etiologies may share a common molecular pathway.     

Manifestations of the neurotoxicity of the organochlorine pesticide dilor during the postnatal period in the rat

When pregnant rats are administered chlororganic pesticide dilor, certain ultrastructural changes are observed in neurocytes and in myelin fibers, in the spinal cord, that demonstrates some disorders in cellular and tissue metabolism. In the newborn spinal cords a retarded differentiation is observed against the background of an intense cellular metabolism. By means of the electron paramagnetic resonance technique, an increasing concentration of free radicals in the brains and spinal cords of the pregnant animals and in their one-month-old offsprings is demonstrated. The investigation on fatty acid composition of lipids in pregnant test animals demonstrates a decrease in cholysterine, steroids and some fatty acids in myelin fractions and in synaptosomic membranes.     

Peroxisomal disorders: complementation analysis using beta-oxidation of very long chain fatty acids

Complementation studies, using fused cell lines from patients with peroxisomal disorders, have shown correction of defective plasmalogen synthesis and phytanic acid oxidation as well as an increase in the number of peroxisomes. At least six complementation groups have been reported. We demonstrate here that complementing cell lines also acquire the ability to oxidize very long chain fatty acids (VLCFA), and that complementation groups defined with this technique are identical to those reported previously when plasmalogen synthesis was used as the criterion for complementation. This VLCFA complementation technique is of particular value in the study of patients in whom defective VLCFA is the only or major enzymatic defect, and we show complementation between cell lines from two patients each with an isolated defect in one of the peroxisomal fatty acid beta-oxidation enzymes.     

Spatial resolution of the variable-period x-ray standing-wave method as applied to model membranes.

A series of model membranes as Langmuir-Blodgett (LB) films composed of long-chain zinc alkanoates (saturated fatty acid salts) was used to evaluate the spatial resolution of the variable-period x-ray standing-wave (XSW) technique. The chain length dependence of the zinc mean position (z) above the supporting substrate demonstrates that it is possible to detect differences in (z) of 1-2 A. Thus 1-2 A is the spatial resolution of the method in the current application. The data show that the chain tilt angle is chain length dependent, varying from 40 degrees to 0 degrees for alkanoates 18 and 24 carbon atoms long, respectively. The spread about the mean position of the zinc in the film, sigma(in), was found to be independent of chain length at 10.0 A for all members of the series. Sigma(in) was shown to be insensitive to the presence of a "spacer" omega-tricosenoic acid (omegaTA) bilayer placed between the zinc alkanoate LB film and the coated gold mirror. However, an overlayer of omegaTA sharpened the zinc ion distribution and lowered the chain tilt angle. This study provides important information regarding sample composition and constitution that facilitates membrane structure determination by XSWs.     

Liquid ionization mass spectrometry of phospholipids

Several phosphatidylcholines (PC) and a phosphatidylethanolamine (PE) were subjected to liquid ionization (LI) mass spectrometry, in which a sample is ionized through energy transfer from metastable argon atoms under atmospheric pressure. Commercially available and synthesized, saturated or unsaturated fatty acid containing phospholipids and their mixtures were studied. A sample either as a concentrated chloroform-methanol solution or with glycerol (matrix) gave characteristic peaks such as MH+ and four fragment ions. One of the fragment ions (e.g., m/z 551 of PC 16:0, 16:0) containing both fatty acid residues has been commonly observed with other ionization methods such as CI, FD, and FAB, but the other fragment ions have not been observed in other mass spectra with one exception on desorption CI. Ions b and d (e.g., m/z 464 and 328, respectively, for PC 16:0, 16:0) contain one fatty acyl residue and the other ion containing the phosphorylcholine moiety appears at m/z 196 for PC. Thus the masses of the MH+ ion and these fragment ions provide useful structural information even in the case of a mixture. The ion b (e.g., m/z 488 of PC 18:0, 18:2) observed during an early period of heating was formed mainly by the loss of one acyl group at sn-1 of the glycerol backbone and thus may be used to differentiate the positional specificities of the constituent fatty acids. The temperature of the sample, however, should be controlled precisely, because it has a significant effect on the mass spectrum. The present method (LI) also provided useful information for a mixture of PC and PE.     

Characterization of fatty amides produced by lipase-catalyzed amidation of multi-hydroxylated fatty acids

Novel multi-hydroxylated primary fatty amides produced by direct amidation of 7,10-dihydroxy-8(E)-octadecenoic acid and 7,10,12-trihydroxy-8(E)-octadecenoic acid were characterized by GC-MS and NMR. The amidation reactions were catalyzed by immobilized Pseudozyma (Candida) antarctica lipase B (Novozym 435) in organic solvent with ammonium carbamate. The mass spectra of the underivatized products exhibited characteristic primary amide peaks at m/z 59 and m/z 72 that differed in peak intensities. Other peaks present were consistent with cleavage next to the hydroxyl groups. The mass spectra of the silylated amidation products showed the correct molecular weight and the typical fragmentation pattern of silylated hydroxy compounds. The mass spectra, together with proton and 13C NMR data, suggest that the products of lipase-catalyzed direct amidation of 7,10-dihydroxy-8(E)-octadecenoic acid and 7,10,12-trihydroxy-8(E)-octadecenoic acid are, 7,10-dihydroxy-8(E)-octadecenamide and 7,10,12-trihydroxy-8(E)-octadecenamide acid, respectively. Amidation of multi-hydroxylated fatty acids had increased the melting point, but reduced the surface active property of the resulting primary amides.     

A radioenzymatic assay to identify three groups of phospholipase A(2) in platelets

Phospholipases A(2) (PLA(2)) are key enzymes in membrane metabolism. The release of fatty acids and lysophospholipids by PLA(2) activates several intra-cellular second messenger cascades that regulate a wide variety of physiological responses. The aim of the present study is to describe a radioenzymatic assay to determine the activity of three main PLA(2) subtypes in platelets, namely extracellular calcium-dependent PLA(2) (sPLA(2)) and intracellular calcium-dependent (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)). The differentiation of these distinct PLA(2) subtypes was based on the enzyme substrate preference (arachdonic acid or palmitoyl acid) and calcium concentration. Our results indicate that this new assay is feasible, precise and specific to measure the activity of the aforementioned subtypes of PLA(2). Therefore, this protocol can be used to investigate modifications of PLA(2) homeostasis in distinct biological models addressing the pathophysiology of many medical and neuropsychiatric disorders such as schizophrenia and Alzheimer's disease.     

Dynamic simulations on the mitochondrial fatty acid Beta-oxidation network

Background  

The oxidation of fatty acids in mitochondria plays an important role in energy metabolism and genetic disorders of this pathway may cause metabolic diseases. Enzyme deficiencies can block the metabolism at defined reactions in the mitochondrion and lead to accumulation of specific substrates causing severe clinical manifestations. Ten of the disorders directly affecting mitochondrial fatty acid oxidation have been well-defined, implicating episodic hypoketotic hypoglycemia provoked by catabolic stress, multiple organ failure, muscle weakness, or hypertrophic cardiomyopathy. Additionally, syndromes of severe maternal illness (HELLP syndrome and AFLP) have been associated with pregnancies carrying a fetus affected by fatty acid oxidation deficiencies. However, little is known about fatty acids kinetics, especially during fasting or exercise when the demand for fatty acid oxidation is increased (catabolic stress).     

Differential subcellular localization of two dopamine D2 receptor isoforms in transfected NG108-15 cells

The dopamine D2 receptor (D2R) is target for antipsychotic drugs and associated with several neuropsychiatric disorders. D2R has a long third cytoplasmic loop and a short carboxyl-terminal cytoplasmic tail. It exists as two alternatively spliced isoforms, termed D2LR and D2SR, which differ in the presence and absence, respectively, of a 29 amino acid insert in the third cytoplasmic loop. To evaluate the differential roles of the two D2R isoforms, we transfected both isoforms into NG108-15 cells and observed their subcellular localization by a confocal laser scanning light microscope. D2SR was predominantly localized at the plasma membrane, whereas D2LR was mostly retained in the perinuclear region around the Golgi apparatus. Using a yeast two hybrid system with a mouse brain cDNA library and coimmunoprecipitation assay, we found that heart-type fatty acid binding protein (H-FABP) interacts with D2LR but not with D2SR. H-FABP is a cytosolic protein involved in binding and transport of fatty acids. Overexpressed H-FABP and endogenous H-FABP were colocalized with the intracellular D2LR in NG108-15 cells. Furthermore, in the rat striatum, H-FABP was detected in the D2R-expressing neurons. From these results, H-FABP is associated with D2LR, and may thereby modulate the subcellular localization and function of D2LR.     

Linoleic acid diols are novel substrates for human UDP-glucuronosyltransferases

Linoleic acid diol glucuronides have been isolated previously from urine of patients suffering from generalized peroxisomal disorders. Glucuronidation of linoleic acid and linoleic acid diols by human liver microsomes was studied to investigate the role of glucuronide conjugation in the metabolism of linoleic acid diols. Glucuronide products were isolated and analyzed by TLC and HPLC-MS. HPLC-MS showed ions with (m/z) corresponding to singly glucuronidated linoleic acid diols while TLC revealed that the glucuronidation was at a hydroxyl position. Kinetic analysis gave apparent K(m) values in the range of 50-200 microM and V(max) rates from 5 to 12 nmol/mg x min. These rates are substantially higher than activities seen for most endogenous hydroxylated substrates. Assays using each of the four individually purified linoleic acid diol enantiomers suggest that glucuronidation occurs at only one of the two hydroxyl groups of each enantiomer. These results show for the first time that hydroxylated fatty acids are actively glucuronidated by human liver microsomes and suggest that glucuronidation may play a significant role in the biotransformation of linoleic acid diols in humans.     

Fatty Acid Composition of Human Myelin Proteolipid Protein in Peroxisomal Disorders

Myelin proteolipid protein (PLP) is an acylated protein which contains approximately 2 mol of ester-bound fatty acids. In this study, the amount and composition of fatty acids covalently bound to human myelin PLP were determined during development and in peroxisomal disorders. Palmitic, oleic, and stearic acids accounted for most of the PLP acyl chains. However, in contrast to PLP in other species, human PLP contains relatively more very long chain fatty acids (VLCFA). The fatty acid composition remained essentially unchanged between 1 day and 74 years of age. The total amount of fatty acid bound to PLP was not altered in any of the pathological cases examined. However, in the peroxisomal disorder adrenoleukodystrophy, the proportions of saturated and, to a lesser extent, monounsaturated VLCFA bound to PLP were increased at the expense of oleic acid. Smaller, but significant, changes were observed in adrenomyeloneuropathy. The reduction in the levels of oleic acid was also observed in two other peroxisomal disorders, the cerebrohepatorenal (Zellweger) syndrome and neonatal adrenoleukodystrophy, as well as in the lysosomal disorder Krabbe globoid cell leukodystrophy. However, in these disorders, the decrease in oleic acid occurred at the expense of stearic acid, and not VLCFA. The results indicate that, although a characteristic PLP fatty acid pattern is normally maintained, changes in the acyl chain pool can ultimately be reflected in the fatty acid composition of the protein. The altered PLP-acyl chain pattern in peroxisomal disorders may contribute to the pathophysiology of these devastating disorders.