Benoit Colsch

Characterization of the ceramide moieties of sphingoglycolipids from mouse brain by ESI-MS/MS identification of ceramides containing sphingadienine

Sphingoglycolipids (SGLs) are cell membrane constituents. As the ceramide structure influences the biological properties of the SGL, we characterized by electrospray ionization tandem mass spectrometry the molecular species of ceramides present in SGL of mouse brain. We report here for the first time the presence in mammalian brain of sphingadienine (d18:2). Sphingenine (d18:1) is present in all SGL species, in contrast to eicosasphingenine (d20:1), which is a constituent of only gangliosides. Sphingadienine is present in galactosylceramide and sulfatides. Free ceramides contain the three types of bases. Thus, there could be two separate pools of free ceramides (d18:1, d18:2 and d20:1, d18:1) as precursors of complex SGL.

Motor and psycho-cognitive clinical types in adult metachromatic leukodystrophy: genotype/phenotype relationships?

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is biochemically characterized by an accumulation of sulfatides (sulfogalactosylceramides) mainly in oligodendrocytes and macrophages/microglia. The deficient enzyme is a lysosomal hydrolase, cerebroside sulfate sulfatase (arylsulfatase A). MLD is both a dysmyelinating and a demyelinating disease. The main clinical forms are infantile or juvenile, but some forms appear at adulthood. This disease involves also neuronal cells as sulfatides are also present in neurons in which the defect in degradation occurs also. We have studied 12 cases of adult MLD and clearly distinguished two clinical forms. One of them was characterized by mainly central nervous system motor signs (pyramidal, cerebellar, and seldom dystonia) and a peripheral neuropathy. The other form always started by behavioural abnormalities with modifications of mood, peculiar social reactions; a progressive mental deterioration occurred also. The diagnosis of schizophrenia was often mentioned. Most of these patients remained for many years without any neurological symptoms, and the diagnosis was only made when neurological signs appeared, or when Magnetic Resonance Imaging (MRI) was performed. MRI showed a diffuse demyelination, bilateral and often symmetrical, which could be temporarily limited to the periventricular areas. The diagnosis of adult MLD was biochemical, evidencing the low activity of arylsulfatase A (ASA) and sulfatide accumulation. To determine the respective participation of neurons and glial cells in the physiopathology of both the motor forms and the psycho-cognitive forms, our first approach was to search for mutations differing according to the clinical status. Motor forms involved the major adult ASA mutation P426L in a homozygote form in contrast to psycho-cognitive forms which involved as a compound heterozygote a specific I179S mutation.

Analysis of free and protein-bound ceramides by tape stripping of stratum corneum from dogs

The free and protein-bound ceramides of dog stratum corneum (SC) were analyzed by thin-layer chromatography after tape stripping of the abdomen of five dogs. The sphingoid bases were identified by gas–liquid chromatography as sphingosine, phytosphingosine, and 6-hydroxysphingosine. Electrospray ionization-ion trap mass spectrometry was used to characterize the protein-bound ceramides containing sphingosine and omega-hydroxy long-chain fatty acids. Although the molecular species were the same ones in all dogs, wide quantitative variations in the patterns of SC ceramides were observed in different breeds of dogs. The free ceramide concentration changed with the depth of SC, with a higher concentration in the deep layers, whereas the concentration of protein-bound ceramides remained constant. These results show that canine SC is close to that of humans with respect to ceramides.

Synthetic sulfogalactosylceramide (sulfatide) and its use for the mass spectrometric quantitative urinary determination in metachromatic leukodystrophies

Abstract3-O-Sulfogalactosylceramides (sulfatides) accumulate in the genetic disease metachromatic leukodystrophy which is due to a defect in the catabolic enzyme, arylsulfatase A. Clinical diagnosis is usually confirmed by in vitro enzymatic deficiency of arylsulfatase A activity. The diagnosis may be complicated because of arylsulfatase A pseudo-deficiencies and another cause of MLD, sphingolipid activator B deficiency. As large quantities of sulfatides can be found in the urine in this disease, sulfatiduria appears as an extremely useful test. As recently enzyme replacement is underway, the quantitative determination, using an internal standard, appears particularly useful as a follow-up. Thus a non-physiological sulfatide was synthesized for this purpose, i.e. 3-O-sulfo-β-d-C17 galactosylceramide (3-O-Sulfo-d-Galactosyl-β1′→1-N-Heptadecanoyl-d-erythro-Sphingosine). It has been prepared through condensation of an azidosphingosine derivative with a protected d-galactopyranosyltrichloroacetimidate. Reduction of the azide was followed by acylation of a C-17 fatty acid. The key step was achieved by selective sulfation of the desired hydroxyl group on the sugar residue of the galactosylceramide using the stannylene methodology to give a 3′-sulfated beta-galactosyl C-17 ceramide.

Mechanistic study of competitive releases of H2O, NH3 and CO2 from deprotonated aspartic and glutamic acids: Role of conformation

The aims of this study were to highlight the impact of minor structural differences (e.g. an aminoacid side chain enlargement by one methylene group), on ion dissociation under collision-induced dissociation conditions, and to determine the underlying chemical mechanisms. Therefore, we compared fragmentations of deprotonated aspartic and glutamic acids generated in negative electrospray ionization. Energy-resolved mass spectrometry breakdown curves were recorded and MS3 experiments performed on an Orbitrap Fusion for high-resolution and high-mass accuracy measurements. Activated fragmentations were performed using both the resonant and non-resonant excitation modes (i.e., CID and HCD, respectively) in order to get complementary information on the competitive and consecutive dissociative pathways. These experiments showed a specific loss of ammonia from the activated aspartate but not from the activated glutamate. We mainly focused on this specific observed loss from aspartate. Two different mechanisms based on intramolecular reactions (similar to those occurring in organic chemistry) were proposed, such as intramolecular elimination (i.e. Ei-like) and nucleophilic substitution (i.e. SNi-like) reactions, respectively, yielding anions as fumarate and α lactone from a particular conformation with the lowest steric hindrance (i.e. with antiperiplanar carboxyl groups). The detected deaminated aspartate anion can then release CO2 as observed in the MS3 experimental spectra. However, quantum calculations did not indicate the formation of such a deaminated aspartate product ion without loss of carbon dioxide. Actually, calculations displayed the double neutral (NH3+CO2) loss as a concomitant pathway (from a particular conformation) with relative high activation energy instead of a consecutive process. This disagreement is apparent since the concomitant pathway may be changed into consecutive dissociations according to the collision energy i.e., at higher collision energy and at lower excitation conditions, respectively. The latter takes place by stabilization of the deaminated aspartate solvated with two residual molecules of water (present in the collision cell). This desolvated anion formed is an α lactone substituted by a methylene carboxylate group. The vibrational excitation acquired by [(D-H)-NH3]-during its isolation is enough to allow its prompt decarboxylation with a barrier lower than 8.4kJ/mol. In addition, study of glutamic acid-like diastereomers constituted by a cyclopropane, hindering any side chain rotation, confirms the impact of the three-dimensional geometry on fragmentation pathways. A significant specific loss of water is only observed for one of these diastereomers. Other experiments, such as stable isotope labeling, need to be performed to elucidate all the observed losses from activated aspartate and glutamate anions. These first mechanistic interpretations enhance understanding of this dissociative pathway and underline the necessity of studying fragmentation of a large number of various compounds to implement properly new algorithms for de novo elucidation of unknown metabolites.

I - 9 Démences de la substance blanche impliquant le métabolisme des lipides

Introduction L’imagerie medicale est une etape importante de l’examen d’un patient dement. L’atteinte de la substance blanche (leucodystrophie), meme isolee, peut etre a l’origine de troubles cognitifs et comportementaux (Filley, 1998). Objectifs Nous avons etudie 8 cas d’adrenoleucodystrophie (ALD) et 8 cas de leucodystrophie metachromatique (LMC) reveles a l’âge adulte, et determine leurs caracteristiques cliniques, neuropsychologiques (tests), et en imagerie par resonance magnetique (IRM). Methodes Le diagnostic a ete etabli, pour l’ALD, par le dosage serique des acides gras a tres longues chaines, et pour la LMC par le dosage d’arylsulfatase A, et par la sulfatidurie qui a ete quantifiee. L’examen neurologique a ete effectue sur une periode de 10 ans. Les tests ont explore le quotient intellectuel, l’attention, les fonctions visuo-spatiales, les memoires declarative et procedurale, le langage, les fonctions du lobe frontal, et le comportement social. Resultats Nous avons observe une absence de troubles du langage et de la memoire procedurale. Il existait des troubles de l’attention, de la memoire declarative, et visuo-spatiaux, et des anomalies du lobe frontal responsables de troubles comportementaux et de l’humeur. Dans la LMC, l’atteinte de la substance blanche etait diffuse, alors que dans l’ALD, la topographie de la demyelinisation entrainait des troubles visuo-spatiaux dans les formes occipitales, des troubles de l’attention dans les formes frontales. Discussion Ces demences different de la maladie d’Alzheimer, car il n’existe ni aphasie, ni agnosie, ni apraxie. Les principaux tests sont axes sur la verbalisation et peuvent donc meconnaitre les alterations specifiques de la substance blanche. La presence de troubles psychiatriques ou comportementaux au premier plan peut faire errer le diagnostic, d’autant que les troubles moteurs sont souvent d’apparition tardive. Conclusion Ces demences de la substance blanche sont liees a des alterations des reseaux neuronaux myelinises. Au debut on peut les reconnaitre ; ulterieurement toutes les fonctions neuro-cognitives sont alterees.