F2-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome

Oxidative damage has been reported in Rett syndrome (RTT), a pervasive developmental disorder caused in up to 95% of cases by mutations in the X-linked methyl-CpG binding protein 2 gene. Herein, we have synthesized F(2)-dihomo-isoprostanes (F(2)-dihomo-IsoPs), peroxidation products from adrenic acid (22:4 n-6), a known component of myelin, and tested the potential value of F(2)-dihomo-IsoPs as a novel disease marker and its relationship with clinical presentation and disease progression. F(2)-dihomo-IsoPs were determined by gas chromatography/negative-ion chemical ionization tandem mass spectrometry. Newly synthesized F(2)-dihomo-IsoP isomers [ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP] were used as reference standards. The measured ions were the product ions at m/z 327 derived from the [M-181](-) precursor ions (m/z 597) produced from both the derivatized ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP. Average plasma F(2)-dihomo-IsoP levels in RTT were about one order of magnitude higher than those in healthy controls, being higher in typical RTT as compared with RTT variants, with a remarkable increase of about two orders of magnitude in patients at the earliest stage of the disease followed by a steady decrease during the natural clinical progression. hese data indicate for the first time that quantification of F(2)-dihomo-IsoPs in plasma represents an early marker of the disease and may provide a better understanding of the pathogenic mechanisms behind the neurological regression in patients with RTT.     

Age-independent,gray matter-localized,brain-enhanced oxidative stress in male fischer 344 rats: brain levels of F(2)-isoprostanes and F(4)-neuroprostanes

While studies showed that aging is accompanied by increased exposure of the brain to oxidative stress, others have not detected any age-correlated differences in levels of markers of oxidative stress. Use of conventional markers of oxidative damage in vivo, which may be formed ex vivo and/or eliminated by endogenous metabolism, may explain these conflicting results. Recently, F₂-isoprostanes and F₄-neuroprostanes, peroxidation products of arachidonic acid and docosahexaenoic acid, respectively, have been identified as sensitive and reliable markers of oxidative injury. Therefore, this study was designed to quantify brain levels of F₂-isoprostanes and F₄-neuroprostanes and their precursors in 4, 10, 50, and 100 week old male Fischer 344 rats. Data show that levels of F₂-isoprostanes and F₄-neuroprostanes were comparable in all animal age groups. However, levels of F₄-neuroprostanes were approximately 20-fold higher than those of F₂-isoprostanes in all age groups, despite the fact that brain levels of docosahexaenoic acid were only twice as high as those of arachidonic acid. Based on our findings, it is concluded that aging is not accompanied by enhanced brain susceptibility to oxidative stress. Furthermore, the metabolically active gray matter of the brain, where docosahexaenoic acid is abundant, appears more susceptible to oxidative stress than the white matter.     

Validating Serum S100B and Neuron-Specific Enolase as Biomarkers for the Human Brain - A Combined Serum, Gene Expression and MRI Study

Introduction

Former studies have investigated the potential of serum biomarkers for diseases affecting the human brain. In particular the glial protein S100B, a neuro- and gliotrophin inducing plasticity, seems to be involved in the pathogenesis and treatment of psychiatric diseases such as major depression and schizophrenia. Neuron-specific enolase (NSE) is a specific serum marker for neuronal damage. However, the specificity of these biomarkers for cell type and brain region has not been investigated in vivo until now.

Methods

We acquired two magnetic resonance imaging parameters sensitive to changes in gray and white matter (T₁-weighted/diffusion tensor imaging) and obtained serum S100B and NSE levels of 41 healthy subjects. Additionally, we analyzed whole brain gene expressions of S100B in another male cohort of three subjects using the Allen Brain Atlas. Furthermore, a female post mortal brain was investigated using double immunofluorescence labelling with oligodendrocyte markers.

Results

We show that S100B is specifically related to white matter structures, namely the corpus callosum, anterior forceps and superior longitudinal fasciculus in female subjects. This effect was observed in fractional anisotropy and radial diffusivity – the latest an indicator of myelin changes. Histological data confirmed a co-localization of S100B with oligodendrocyte markers in the human corpus callosum. S100B was most abundantly expressed in the corpus callosum according to the whole genome Allen Human Brain Atlas. In addition, NSE was related to gray matter structures, namely the amygdala. This effect was detected across sexes.

Conclusion

Our data demonstrates a very high S100B expression in white matter tracts, in particular in human corpus callosum. Our study is the first in vivo study validating the specificity of the glial marker S100B for the human brain, and supporting the assumption that radial diffusivity represents a myelin marker. Our results open a new perspective for future studies investigating major neuropsychiatric disorders.     

STALOSYLGALACTOSYL CERAMTDE AS A SPECIFIC MARKER FOR HUMAN MYELIN AND OLIGODENDROGLIAL PERIKARYA: GANGLIOSIDES OF HUMAN MYELIN, OLIGODENDROGLIA AND NEURONS

Gangliosides were isolated from human brain myelin, oligodendroglia, and neurons. Quantitative analysis revealed the following ganglioside contents: myelin, 2.0; neurons, 1.3; and oligodendroglia, 0.35 μg ganglioside sialic acid per mg protein. Myclin had a relatively simple ganglioside pattern with G_M4 and G_M1 as the predominant ganglioside species. The ganglioside pattern of oligodendroglia was quite complex and it resembled that of whole white matter rather than that of myelin. A high concentration of G_M4 was found in oligodendroglial fractions in addition to G_M1, G_D1a, G_D1b, and G_T1b. The usually- minor brain gangliosides G_M3, G_M2, and G_M3 were also enriched in oligodendroglia. The neuronal ganglioside pattern was generally similar to the pattern of whole gray matter. Both neurons and whole gray matter contained very low amounts of G_M4. These results indicate that G_M4 is specifically localized in myelin and oligodendroglia of the CNS. Evidence is also presented that myelin, but not oligodendroglia, is the major reservoir of human white matter G_M1 and G_M4.     

Identification of Plasmolipin as a Major Constituent of White Matter Clathrin-Coated Vesicles

We have isolated and characterized coated vesicles from bovine white matter and compared them to those isolated from gray matter. The virtual absence of synaptic vesicle antigens in the white matter coated vesicles indicates they are distinct from those found in gray matter and from vesicles derived from synaptic membranes. The white matter coated vesicles also lack compact myelin components, e.g., the myelin proteolipid, galactocerebroside, and sulfatides, as well as the periaxolemmal myelin marker myelin-associated glycoprotein. On the other hand, these vesicles contain 2',3'-cyclic nucleotide phosphohydrolase. The vesicles also contain high levels of plasmolipin, a protein present in myelin and oligodendrocytes. Plasmolipin was found to be four to five times higher in white matter coated vesicles than in gray matter coated vesicles. Based on western blot quantitation, the concentration of plasmolipin in white matter coated vesicles is 3-4% of the vesicle bilayer protein. These studies indicate that a significant proportion of coated vesicles from white matter may be derived from unique membrane domains of the myelin complex or oligodendroglial membrane, which are enriched in plasmolipin.     

Phospholipids and acyl groups in subcellular fractions from human cerebral cortex

Subcellular fractionation of human brain cortex obtained at autopsy yielded microsomal and synaptosome-rich fractions from the gray matter and microsomal and purified myelin fractions from the white matter. The phospholipids of myelin were high in plasmalogens, and the molar ratio of alkenyl acyl sn-glycero-3-phosphorylethanolamine to diacyl sn-glycero-3-phosphorylethanolamine was 4. The acyl groups of the myelin phosphoglycerides were enriched in monoenes (mainly 18:1 and 20:1) and a tetraene, 22:4(n - 6). The phospholipids in the synaptosome-rich fraction were high in diacyl sn-glycero-3-phosphorylcholine, and the molar ratio of the alkenyl acyl sn-glycero-3-phosphorylethanolamine to diacyl sn-glycero-3-phosphorylethanolamine was 0.88. The acyl groups of synaptosomal ethanolamine phosphoglycerides were rich in 22:6(n - 3) but contained a very low amount of 20:1. The lipid composition of microsomes from the gray matter was different from that of microsomes from the white matter but was nearly identical with that of the synaptosome-rich fraction. Except for a slightly lower proportion of alkenyl acyl sn-glycero-3-phosphorylethanolamine and sphingomyelin, the lipid composition of microsomes from the white matter was also similar to that of the myelin. There were also species-related differences between the brain lipid composition of human and subhuman primates and that of the rodents. Furthermore, the brain lipid composition in normal human subjects is rather constant and does not seem to be affected much by individual variations.     

Neuronal oxidative damage from activated innate immunity is EP2 receptor-dependent

Increase in prostaglandin (PG) E2 levels and oxidative damage are associated with diseases of brain that involve activation of innate immunity. We tested the hypothesis that cerebral oxidative damage resulting from activation of innate immunity with intracerebroventricular (icv) lipopolysaccharide (LPS) is dependent on PGE2-mediated signaling. We measured two quantitative in vivo biomarkers of lipid peroxidation: F2-isoprostanes (IsoPs) that derive from arachidonic acid (AA) that is uniformly distributed in all cell types in brain, and F4-neuroprostanes (NeuroPs) that derive from docosahexaenoic acid (DHA) that is highly concentrated in neuronal membranes. LPS stimulated delayed elevations in cerebral F2-IsoPs and F4-NeuroPs that were completely suppressed by indomethacin or ibuprofen pre-treatment. LPS-induced cerebral oxidative damage was abolished by disruption of subtype 2 receptor for PGE2 (EP2). In contrast, initial oxidative damage from icv kainic acid (KA) was more rapid than with LPS also was completely suppressed by indomethacin or ibuprofen pre-treatment but was independent of EP2 receptor activation. The protective effect of deleting the EP2 receptor was not associated with changes in cerebral eicosaniod production, but was partially related to reduced induction of nitric oxide synthase (NOS) activity. These results suggest the EP2 receptor as a therapeutic target to limit oxidative damage from activation of innate immunity in cerebrum.     

Rat brain fatty acid-binding protein during development.

Cytosolic fatty acid-binding proteins (FABPs) have been described in rat and bovine whole brain. In the present study we investigated the distribution of FABP among white matter and gray matter as well as its changes during development. Fatty acid binding activity was similar in white and gray matter up to 40 days of age. In white matter it showed an age dependent increase thereafter, while in gray matter it remained constant throughout. Gel filtration (Sephadex G-75) of white matter cytosol of adult female rats resolved the fatty acid-binding activity in two peaks: A (Vo) and B (12-14 KDa; FABP). The specific binding activity in the FABP fraction was 10.4 pmol/micrograms of protein. The activity in peak A showed an age-dependent increase which paralleled myelin deposition. In contrast, the activity in the FABP fraction (peak B) remained undetectable up to 40 days of age, increasing thereafter. The differential distribution of cellular brain proteins with the capacity to bind fatty acids in gray matter and white matter suggests that this activity could be related to glial cells or to cell related structures such as myelin.     

Gray matter imaging in multiple sclerosis: what have we learned?

At the early onset of the 20^th century, several studies already reported that the gray matter was implicated in the histopathology of multiple sclerosis (MS). However, as white matter pathology long received predominant attention in this disease, and histological staining techniques for detecting myelin in the gray matter were suboptimal, it was not until the beginning of the 21^st century that the true extent and importance of gray matter pathology in MS was finally recognized. Gray matter damage was shown to be frequent and extensive, and more pronounced in the progressive disease phases. Several studies subsequently demonstrated that the histopathology of gray matter lesions differs from that of white matter lesions. Unfortunately, imaging of pathology in gray matter structures proved to be difficult, especially when using conventional magnetic resonance imaging (MRI) techniques. However, with the recent introduction of several more advanced MRI techniques, the detection of cortical and subcortical damage in MS has considerably improved. This has important consequences for studying the clinical correlates of gray matter damage. In this review, we provide an overview of what has been learned about imaging of gray matter damage in MS, and offer a brief perspective with regards to future developments in this field.     

Determination of salicylate hydroxylation products as an in vivo oxidative stress marker

The in vivo measurement of highly reactive free radicals, such as the z.rad OH radical, is very difficult. New specific markers, which are based on the ability of z.rad OH to attack the benzene rings of aromatic molecules, are currently under investigation. The produced hydroxylated compounds can be measured directly. In vivo, radical metabolism of salicylic acid produces two main hydroxylated derivatives (2,3- and 2,5-dihydroxybenzoic acids). The latter acid can be also produced by enzymatic pathways through the cytochrome P-450 system, while the former acid is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2, 3-DHBA, following oral administration of the drug acetyl salicylate, could be proposed for assessment of oxidative stress in vivo. In this paper, a sensitive method for the identification and quantification of hydroxylation products from the reaction of z. rad OH with salicylate in vivo is presented. It employs a high performance liquid chromatography and electrochemical detection system. A detection limit of < 1 pmol for the hydroxylation products has been achieved with linear response over at least five orders of magnitude. Using this technique, we measured plasma levels of 2,3- and 2,5-DHBA dihydroxylated derivatives and salicylic acid and determined the ratios following administration of 1 g acetyl salicylate in 20 healthy subjects.     

Effects of Neonatal Undernutrition on the Lipid Composition of Gray Matter and White Matter in Rat Brain

Abstract: Separate analyses were made of gray matter and white matter from rat brain after neonatal undernutrition. Newborn rats were redistributed into control, large-litter, and protein-deficient groups. Large litters had 16 rather than 8 pups with a dam. Protein-deficient dams were fed a 4%, instead of a 24%, casein diet. For controls at 21 days of age, the 2',3'-cyclic nucleotide-3'-phosphohydrolase activity was more than fivefold greater in white matter than in gray matter. Severe undernutrition (protein-deficient) gave 2',3'-cyclic nucleotide-3'-phosphohydrolase activities that were 36% lower in gray matter and 56% lower in white matter. Lipid galactose concentrations were 17% less than control in both gray matter and white matter. In protein-deficient white matter, phospholipid concentrations were 15% lower than control. Ethanolamine plasmalogens and phosphatidyl serine were affected most. Moderate undernutrition (large litter) had no effect on 2',3'-cyclic nucleotide-3'-phosphohydrolase activity. A 14% deficit of galactolipids was the only difference from controls in large-litter white matter. In large-litter gray matter, phospholipid concentrations were 16% higher than controls. Nearly all glycerophos-pholipids, including plasmalogens, were affected. With the exception of the myelination markers, 2',3'-cyclic nucleotide-3'-phosphohydrolase and lipid galactose, the development of lipids in gray matter is almost completely spared from the effects of undernutrition. The primary effect of undernutrition is on myelination, especially in white matter.     

ATP-dependent adenophostin activation of inositol 1,4,5-trisphosphate receptor channel gating: kinetic implications for the durations of calcium puffs in cells

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) is a ligand-gated intracellular Ca(2+) release channel that plays a central role in modulating cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP(3)R that is structurally different from InsP(3) and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP(3)R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP(3)R activated by either AdA or InsP(3) have identical channel conductance properties. Furthermore, AdA, like InsP(3), activates the channel by tuning Ca(2+) inhibition of gating. However, gating of the AdA-liganded InsP(3)R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP(3)-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP(3) in the presence of 0.5 mM ATP, although the functional affinity of the channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP(3)R were very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probability than channels activated by AdA in the presence of ATP, or by InsP(3) in the presence or absence of ATP. Also, the higher functional affinity of InsP(3)R for AdA than for InsP(3) is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP(3)R channels with gating properties similar to those of AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, including the mechanisms that determine the durations of elementary Ca(2+) release events in cells. Comparisons of single-channel gating kinetics of the InsP(3)R activated by InsP(3), AdA, and its analogues also identify molecular elements in InsP(3)R ligands that contribute to binding and activation of channel gating.     

ACYL AND ALK-1'-ENYL GROUP COMPOSITION OF ETHANOLAMINE PHOSPHOGLYCERIDES OF HUMAN BRAIN

Ethanolamine phosphogylcerides (EPG) of human brain gray and white matter were analyzed for their alk-1′-enyl group and fatty acid compositions in sn-glycerol positions 1 and 2. Gray matter contained more 18:0 (54%) and less 18:1 (24.5%) alk-1′-enyl residues than white matter (16% and 57%. Sixty per cent of alk-1′-enyl 18.1 in gray matter was the (n-7), against 71%, in white matter. Both gray and white matter contained small amounts of 18:1 (n-5) and (n-3) isomers. The fatty acids in position I of the phosphatidylethanolamines were more saturated than the corresponding alk-1′-enyl groups of the plasmalogens. The ratios of monoenoic fatty acid isomers in position 1 were markedly different from those of the corresponding alk-1′-enyl groups in gray matter. The fatty acid patterns in position 2 of plasmalogen and phosphatidylethanolamines of white matter were similar except for 22:4(n-6) which was concentrated in the plasmalogen (16% against 10%, in the phosphatidyl ethanolamine). In gray matter, the same trend was noted. The data suggest that alk-1′-enyl residues and the fatty acids in position 1 as well as the fatty acids in position 2 of alk-1′-enyl acyl and diacyl type EPG in both gray and white matter are, at least in part, of different provenance.     

Changes in fatty acid composition of human brain myelin lipids during maturation

Abstract— The variation with age of the fatty acid composition of the major lipids in human brain myelin was compared with that of cerebral white matter from the same region. The myelin was isolated from the semiovale centre of the cerebrum of 27 subjects neonatal to old aged. The phospholipid, cholesterol and galactolipid concentrations were determined in all the samples, as were the proportions of the major phospholipid classes. The proportions of cholesterol and especially of the galactolipids increased in myelin during the first 6 months, and in cerebral white matter up to 2 years. During this period the individual phospholipids also varied substantially. Serine phosphoglycerides and especially sphingomyelins increased, and choline phosphoglycerides decreased. The fatty acid patterns of ethanolamine phosphoglycerides (EPG) and sphingomyelins underwent the largest changes. The proportions of saturated fatty acids in EPG diminished rapidly, and there was an increase of monoenoic acids. Fatty acids of the linoleic acid series showed a peak between 4 and 12 months, after which time their proportion slowly diminished to old age. The major fatty acid of this series was docosatetraenoic acid, 22:4 (n-6), which constituted more than 25% of total fatty acids at the maximum level. The fatty acid changes were larger in cerebral white matter, but from 2 years of age the EPG fatty acid pattern in myelin was similar to that in white matter. The fatty acid changes in serine and choline phosphoglycerides of myelin with maturation were much less striking than in EPG but of a similar type. In myelin sphingomyelin the proportion of saturated long-chain fatty acids, C₁₆-C₂₂, diminished, while that of monoenoic acids increased and continued to do so up to old age. From 2 years of age the fatty acid patterns in myelin and cerebral white matter were quite similar. Also the fatty acid patterns of cerebrosides and sulphatides in cerebral white matter and myelin were the same except for the first 2 months of life. The same fatty acid changes occurred in cerebrosides and sulphatides as in the sphingomyelins, i.e. increased proportions of unsaturated (monoenoic) acids. The proportions of 24:1 and 24h:1 and of the odd-numbered fatty acids 25:1 and 23h:1 continued to increase to old age. The variations of the individual lipid fatty acid patterns were small except in the youngest age classes, in which the variations were presumably ascribable to the difficulty in determining the gestational age.     

Regional Central Nervous System Oligosaccharide Storage in Caprine β-Mannosidosis

Goats affected with beta-mannosidosis, an autosomal recessive disease of glycoprotein metabolism, have deficient activity of the lysosomal enzyme beta-mannosidase along with tissue storage of oligosaccharides, including a trisaccharide [Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc] and a disaccharide [Man(beta 1-4)GlcNAc]. CNS myelin deficiency, with regional variation in severity, is a major pathological characteristic of affected goats. This study was designed to investigate regional CNS differences in oligosaccharide accumulation to assess the extent of correlation between oligosaccharide accumulation and severity of myelin deficits. The concentrations of accumulated disaccharide and trisaccharide and the activity of beta-mannosidase were determined in cerebral hemisphere gray and white matter and in spinal cord from three affected and two control neonatal goats. In affected goats, the content of trisaccharide and disaccharide in spinal cord (moderate myelin deficiency) was similar to or greater than that in cerebral hemispheres (severe myelin deficiency). Thus, greater oligosaccharide accumulation was not associated with more severe myelin deficiency. Regional beta-mannosidase activity levels in control goats were consistent with the affected goat oligosaccharide accumulation pattern. The similarity of trisaccharide and disaccharide content in cerebral hemisphere gray and white matter suggested that lysosomal storage vacuoles, more numerous in gray matter, may not be the only location of stored CNS oligosaccharides.     

Soluble and bound acid protease activity of myelin from bovine cerebral white matter and spinal cord

Isolated myelin of bovine spinal cord was found to degrade exogenous myelin basic protein (MBP) at pH 4.4. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Some of the proteolytic activity was soluble at increased ionic strength, some remained bound, withstanding extraction at 37°C for up to 12 hr. While being measurable with exogenous MBP, bound protease degraded neither bound MBP nor any other major intrinsic myelin protein. Both soluble and bound protease activity was completely inhibited by pepstatin A. The patterns of limited proteolysis of MBP they produced were identical. Myelin of cerebral white matter also exhibited soluble and bound acid protease activity which was likewise inhibited by pepstatin A. Protease activity of spinal cord and cerebral myelin is therefore suggested to be due to a cathepsin D-like endopeptidase, present in a loosely and tightly bound form. Both forms increased by 50 to 80% in activity when myelin was isolated from mixtures of white and cortical gray matter. While increased soluble activity of myelin is consistent with binding of cathepsin D of lysosomal origin during the isolation of myelin the tightly bound form might point to a principal mechanism through which exogenous proteins may become attached to the myelin sheath in vivo.     

Disorder in human myelin induced by superoxide radical: an in vitro investigation

Potassium superoxide (KO.2), applied as a source of superoxide radical directly in vitro to white matter from young adult human brain, caused the lipid phase of the myelin to change from a crystalline (ordered) state to a liquid crystalline (disordered) state. The myelin transition temperature decreased from 65 degrees C to 37 degrees C. This alteration was accompanied by a dramatic increase in the levels of lipid peroxidation products--malondialdehyde, a conjugated diene, and ethane. These changes in human myelin, induced by direct application of O2-. radical, simulated myelin deterioration that occurs in the course of natural aging, thus, providing further substantiation for the notion that O2-. might be a major toxic agent associated with the aging process.     

Histochemical visualization of oxidant stress

Free radicals induce oxidative modification in distinct components of the living matter (lipid, proteins, and DNA). For qualitative and quantitative determination of free radical-induced modifications, different, more or less sensitive biochemical methods are available. Because of the high reactivity and short life of free radicals, ongoing oxidative damage is generally analyzed by measurement of secondary products-such as H(2)O(2), oxidized proteins, peroxidized lipids, and their breakdown products, oxidized DNA-or by fluorographic analysis in combination with fluorescent dyes such as dichlorofluorescin (DCFH). In addition, the determination of free radical-related oxidation products is usually carried out in plasma, urine, or, less frequently, in bioptic material. Consequently, biochemical data seldom reflect the effects of free radical insults in situ. The histochemical visualization of selected molecular markers of oxidative damage can often provide more valuable information concerning the in vivo distribution of oxidative processes. This review summarizes the methods currently available for histochemical detection and indirect visualization of free radical-induced alterations in tissues and isolated cells.     

Binding of delta-aminolevulinic acid by myelin proteins

Delta-[14C]aminolevulinic acid (ALA) was injected intracranially into experimental animals; the gray and white matter was obtained from the brain 24 hours thereafter. The radioactive label content in the ALA, protoporphyrin, microsomes, mitochondria, cell membranes and myelin was determined; no radioactivity was detected in ALA and protoporphyrin. The radioactive label was localized in the subcellular fractions and myelin, in particular, in the white matter. Analysis of protein myelin fractions demonstrated that ALA was incorporated into practically all basic proteins. The highest capacity to bind ALA was observed in case of Wolfgram proteins; however, almost half of the bound ALA in the myelin fraction was found within the composition of basic proteins. It was assumed that the binding of ALA to proteins occurs via the amide, carboxylic and keto groups of ALA.     

Phospholipid sources for adrenic acid mobilization in RAW 264.7 macrophages. Comparison with arachidonic acid

Cells metabolize arachidonic acid (AA) to adrenic acid (AdA) via 2-carbon elongation reactions. Like AA, AdA can be converted into multiple oxygenated metabolites, with important roles in various physiological and pathophysiological processes. However, in contrast to AA, there is virtually no information on how the cells regulate the availability of free AdA for conversion into bioactive products. We have used a comparative lipidomic approach with both gas chromatography and liquid chromatography coupled to mass spectrometry to characterize changes in the levels of AA- and AdA-containing phospholipid species in RAW 264.7 macrophage-like cells. Incubation of the cells with AA results in an extensive conversion to AdA but both fatty acids do not compete with each other for esterification into phospholipids. AdA but not AA, shows preference for incorporation into phospholipids containing stearic acid at the sn-1 position. After stimulation of the cells with zymosan, both AA and AdA are released in large quantities, albeit AA is released to a greater extent. Finally, a variety of phosphatidylcholine and phosphatidylinositol molecular species contribute to AA; however, AdA is liberated exclusively from phosphatidylcholine species. Collectively, these results identify significant differences in the cellular utilization of AA and AdA by the macrophages, suggesting non-redundant biological actions for these two fatty acids.