Hildegard Debuch

2′, 3′‐Cyclic Nucleotide 3′‐Phosphohydrolase and Lipids of Myelin from Multiple Sclerosis and Normal Brains

Abstract: A study of purified myelin samples from normal‐appearing white matter of 10 multiple sclerosis (MS) brains was undertaken and the results were compared with 10 age‐matched control brains. Statistical evaluations were carried out with Students r‐test for differences. In pathological samples the yield of myelin came to only two‐thirds of the corresponding controls. Enzyme assays of the 2′, 3′‐cyclic 3′‐phosphohydrolase revealed an obviously significant reduction of specific activity to one‐half in MS myelins. In myelin the contents of protein, lipid classes as cholesterol, glycolipids and phospholipids did not differ significantly. No cholesterol esters or any lysophospholipid were detectable either in MS or in controls. Within the individual phospholipids the main components were in the same order, while a significant decrease of the acidic representatives and of sphingomyelin occurred. Analysis of the fatty acid pattern of phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), including the aldehydes from the last, revealed quite similar values with no significant differences, except C22: 4 fatty acid in the PE fraction and C20: 1 fatty acid in PS, which were reduced in MS myelin samples.

Lysoplasmalogenase–A Microsomal Enzyme from Rat Brain

Abstract: An enzymic activity of rat brain that liberates radioactive free aldehydes from 1‐[1‐14C]alk‐1′‐enyl‐sn‐glycero‐3‐phosphoethanolamine (lysoplasmalogen) is described. It was present mainly in microsomal fractions (crude) of brains of rats of different ages. The highest specific enzyme activity was found in 21‐day‐old animals. The formation of free aldehyde was dependent on the amount of enzyme protein as well as the amount of substrate added, and was linear to the incubation time up to 60 min. The pH optimum was between 7.1 and 7.3. Bivalent cations (Mg2+, Ca2+) and detergents inhibited the reaction. However, the same cell fractions as well as extracts of acetone‐dried powder of brain from young or old rats possessed no enzyme activity for liberating the aldehyde from the acylated substrates: 1‐[1‐14C]alk‐1′‐enyl‐2‐acyl‐sn‐glycero‐3‐phosphoethanolamine (plasmalogen) or plasmalogen of ox corpus callosum.

Evaluation of a gas chromatographic method for the quantitative estimation of hexoses from neutral glycolipids

Abstract The alditol acetate method for the estimation of the hexoses from the carbohydrate moiety in neutral glycolipids has been standardized using gas–liquid chromatographic conditions which provided better resolution of the individual neutral sugars. The method involves the aqueous acid hydrolysis of the glycolipid, reduction of the released free sugars to the alcohols and acetylation before gas chromatographic analysis. Glucose-containing lipids differ from galactolipids in that the former require a longer time of hydrolysis for a quantitative cleavage of the sugar from the ceramide moiety. Results of the application of this method to glycolipids in the range of 0.2 to 0.5 μmole gave quantitative values using arabinose as internal standard. The sugar estimations of cerebrosides and sulphatides in the presence of phosphatides also gave quantitative results. The method has also been applied to determine the galactose to glucose ratio of ceramide di-, tri- and tetrahexosides of human erythrocyte ghost lipids.

Untersuchungen an Glycerinphospholipiden aus weißer Substanz von MS- und Normalgehirnen

SummaryIn order to determine the total lipid content, as well as the fatty acid and aldehyde content of the two principal glycerophospholipid fractions, the cholinephosphatides and the ethanolaminephosphatides, 17 samples of normal looking white matter from various regions of four brains of MS patients, and 21 samples from the corresponding regions of five normal brains, were analyzed. The MS patients had suffered from the disease for 8 to 20 years and the diagnosis had been established histologically.A statistically significant difference, expressed in mg/g of dry tissue weight, was found in the normal looking white matter of the MS brains compared with that of the normal brains, as follows:(1)The lipids in general and every lipidfraction itself (total cholesterol, phospholipids and glycolipids) were reduced.(2)The reduction of the different phospholipid families varied. The decrease of ethanolamineplasmalogens and of phosphatidylserines was the greatest (25%), whereas the remaining ethanolamine phospholipids and the phosphatidylcholines were nearly the same as in normal tissue.(3)Fatty acids of phosphatidylcholines showed a decrease of C18:0 and C18:1. The absolute amount of palmitic acid was by no means reduced, but seemed to be increased.(4)The fatty acids, a well as the aldehydes of the ethanolamine phospholipids, showed a decrease of C18:1. The other aldehydes were also diminished, as was also true for the C20:1 fatty acid. We think it important that there was a difference in the amount of reduction in the phospholipid families and a decrease of the monounsaturated compounds in the main glycerophospholipids.ZusammenfassungAus 4 MS-Gehirnen entnahmen wir 17 Proben von makroskopisch unveränderter weißer Substanz und verglichen sie hinsichtlich der Gesamtlipide, der Phospholipidzusammensetzung sowie der Fettsäuren und Aldehyde aus den cholin- bzw. ethanolaminhaltigen Phospholipiden mit 21 entsprechenden Proben aus 5 Normalgehirnen. Die weiße Substanz stammte vor allem aus den großen Marklagern des Frontal-, Parietal-, Occipital-Hirns und einiger anderer Regionen. Die Krankheitsdauer der Patienten betrug 8–20 Jahre. Die Diagnose war durch histologische Kontrollen gesichert.Folgende Ergebnisse, gewonnen durch die Untersuchung der „normal“ erscheinenden weißen Substanz der MS-Gehirne, waren hoch signifikant — (angegeben in mg/g getr. Gewebe).1.Die Gesamtlipide und die einzelnen Lipidfraktionen (Gesamtcholesterin, Phospholipide und Glycolipide) waren vermindert.2.Die Abnahme der verschiedenen Phospholipidklassen war sehr unterschiedlich. Dabei zeigten die Ethanolaminplasmalogene und die serinhaltigen Phospholipide die stärkste Reduktion (etwa um 25%), während die übrigen Ethanolaminphospholipide im Material aus pathologischen Gehirnen in gleicher Menge wie in den normalen Proben vorlag. Das Phosphatidylcholin wies nur eine Abnahme von 5% auf.3.Die Fettsäuren des Phosphatidylcholins zeigten eine Abnahme der C18:0-und C18:1-Verbindungen. Die absolute Menge der C16:0-Fettsäure war keineswegs vermindert. Sie war — allerdings nicht signifikant — angestiegen.4.Sowohl bei den Fettsäuren als auch den Aldehyden der ethanolaminhaltigen Phospholipide hatten die C18:1-Verbindungen stark abgenommen. Bei den Aldehyden waren auch die anderen Komponenten betroffen, bei den Fettsäuren besonders auch die C20-Monoene. Die spezifische Verminderung einiger Glycerinphospholipide wie auch die mehrfach anzutreffende Abnahme der Monoenverbindungen erscheint uns besonders bedeutsam.

Nature of the linkage of the aldehyde residue of natural plasmalogens.

FEULCEN and VOIT (1924) first demonstrated with tissue slices that the cytoplasm gave an aldehyde reaction with fuchsin-sulphurous acid. FEULGEN and his coworkers (1929) were able to isolate the aldehydes (called plasmals, because they occurred in the plasma of the cells) and identified them as a mixture of palmital and stearal; finally, FEULGEN and BERSIN (1939) separated a phospholipid from an extract of beef heart muscle which contained these aldehydes linked as an acetal. To this acetalphospholipid (or plasmalogen, because it produced the aldehydes (plasmals) after acid treatment) they ascribed the following formula :

Lipid investigation of central and peripheral nervous system in connatal Pelizaeus-Merzbacher's disease.

Abstract: Brain grey and white matter of a case of Pelizaeus‐Merzbacher disease (connatal type Seitelberger) of a 19‐month‐old boy were analysed with respect to lipids. Cerebrosides and sulfatides were totally absent in the pathological brain. In comparison to control, differences in gangliosides could be detected in grey and white manner. C18:1, fatty acids were markedly reduced in the main glycerophospholipids of white matter. In sphingomyelin of cortex and white matter 90% of fatty acids were C18:0; longer chains were absent. In contrast: PNS (nervus fernoralis) lipids contained the main galactolipids. However, these a s well as all other lipid classes showed a 20% reduction compared with values obtained from nervus femoralis of an infant of the same age. The fatty acid patterns of all lipid classes were determined. The only marked deviations from normal were observed in the C24‐chains of cere‐brosides and sulfatides. The formalin‐fixed brain of an older brother (same disease) was analysed only with respect to glycolipids: neither cerebrosides nor sulfatides could be detected.

On the Phospholipid Metabolism of Glial Cell Primary Cultures: Cell Characterization and Their Utilization of 1-Alkyl-Glycerophosphoethanolamine

Abstract: Primary cultures prepared from newborn rat brain were grown for 16 or 17 days in culture. Addition of brain extract from newborn rats to the medium stimulated the maturation of astrocytes and the development of oligodendrocytes. Both cell types were characterized by morphology and by immunohistochemistry. The phospholipid composition of these cells was estimated. Incubations were performed with l‐[3H]alkyl‐sn‐glycerophosphoethanolamine in varying concentrations for 3 h. About one‐third of the substrate supplied was internalized by the cells. Several enzymic reactions were observed. The acylating enzyme system was the most active one—a Km was determined with 5 nmol intracellular 1‐alkyl‐sn‐glycerophosphoethanolamine/mg cell protein. Plasmalogen formation was rather low. 1‐Alkyl‐sn‐glycerol, a hydrolysis product, was found in small amounts. Some radioactivity was also incorporated into the phosphatidylcholine fraction.

Influence of chloroquine treatment on enzymes and phospholipids from rat liver cell fractions

Abstract Rats, treated for 12 days with chloroquine show a threefold increase of arylsulfatase activity in the mitochondrial-lysosomal mixed fraction, whereas the succinate: cytochrome c reductase activity is decreased to about 50% in this fraction. Purified lysosomes possess a 35 fold higher arylsulfatase activity, compared with homogenate, whereas neither NADPH: - nor succinate: cytochrome c reductase activity can be detected. In these lysosomes, one third of the phospholipids consists of bis (monoacylglycero) phosphate. The neutral phospholipids — mainly phosphatidylethanolamine — are drastically reduced in these cell organelles during the treatment. Our results indicate that chloroquine is nearly exclusively present in the lysosomal fraction. Furthermore we conclude from our data that bis (monoacylglycero) phosphate — isolated from lysosomal phospholipids — forms complexes with chloroquine.

Alkenylhydrolase: a microsomal enzyme activity in rat brain

Abstract: Rat brain microsomes have the capacity to liberate radioactive free aldehydes from 1‐[1‐14C]alk‐1′‐enyl‐sn‐glycero‐3‐phosphoethanolamine (lysoplasmalogen). Glycerophosphoethanolamine was found using 1‐alk‐1′‐enyl‐sn‐glycero‐3‐phospho‐[3H]ethanolamine. The ratio of both products released by lysoplasmalogenase action was 1:1. Another enzymic activity could be demonstrated, which hydrolyzes lysoplasmalogen at the hydrophilic part of the molecule, a lysophospholipid phospho diesterase. Thus, 1‐[1‐14C]alk‐1′‐enylglycerol was detected as well as [3H]ethanolamine, again in a molar ratio, from the respective labeled substrates. This enzyme possesses nearly the same affinity toward the substrate as lysoplasmalogenase. Whereas the lysophospholipid phos phodiesterase is totally inhibited in the presence of NaF or EDTA, lysoplasmalogenase activity is not affected by these reagents. 1‐[1‐‘14C]Alk‐l’‐enylglycerol acts also as substrate for lysoplasmalogenase, which liberates radioactive aldehydes at the same rate as from lysoplasmal ogen. Because the apparent Km and Vmax values are nearly identical for both substrates, the enzyme activities are inhibited in the same way, and the pH optimum is about 7.2 in both cases, it is concluded that both substrates were attacked by the same enzyme. The enzyme does not differentiate between a substrate substituted at the sn‐3 position of glycerol and one that is not. It requires only a free OH group at the sn‐2 position. Phosphoethanolamine phosphatase activity was also determined under our experimental conditions.

Lymph node excision as a simple diagnostic acid in rare lipidoses

Autopsy material from a case of Niemann-Pick disease was subjected to lipid analysis. Among the six tissues investigated, lymph nodes exhibited the greatest storage of several lipids. Since lymph nodes are relatively easy to obtain by biopsy, they may be utilized for chemical diagnosis of this type of lipidosis.