Helmut Woelk

THE SYNTHESIS OF CHOLINE AND ETHANOLAMINE PHOSPHOGLYCERIDES IN NEURONAL AND GLIAL CELLS OF RABBIT IN VITRO

Abstract— The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labelled phosphorylcholine (phosphorylethanolamine) or cytidine‐5′‐phosphate choline (cytidine‐5′‐phosphate ethanolamine), as lipid precursors. Synthesis of phospholipid from phosphorylcholine and phosphorylethanolamine in both fractions was extremely low when compared to that derived from the corresponding cytidine nucleotides. The neuronal cell‐enriched fraction was found to possess a much higher rate of synthesis of both lipids from all precursors. Neuronal/glial ratios of about 5–9 were found for the synthesis of phosphatidylcholine and phosphatidylethanolamine from cytidine‐5′‐phosphate choline and cytidine‐5′‐phosphate ethanolamine, respectively. Several kinetic properties of the choline‐phosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found to be similar both in neurons and in glia (e.g. Km of cytidine‐5′‐phosphate ethanolamine, Km of diacyl glycerol, pH optimum, need for divalent cations), but the Km value for cytidine‐5′‐phosphate choline in glial cells was much lower (2.3 × 10−4m) than in neurons (1 × 10−3m). The Kmfor cytidine‐5′‐phosphate ethanolamine in both cells was much lower than in whole brain microsomes. It is concluded that the cytidine‐dependent enzymic system for phosphatidylcholine and phosphatidylethanolamine synthesis is concentrated mostly in the neuronal cells, as compared to glia.

Lipid and Fatty Acid Composition of Myelin Purified from Normal and MS Brains

The concentrations of phospholipids and galactolipids and their fatty acid compositions were determined in purified myelin from apparently normal white matter of 11 multiple sclerosis (MS) and 11 control brains. Myelin from MS brains showed a significant decrease in serine phosphatide and a slight decrease in the ethanolamine phosphatide fraction when compared with normal myelin; increases were found in the lysocholine and lysoethanolamine phosphoglycerides. In the galactolipids, lower figures were obtained for sulfatides, resulting in a higher cerebroside/sulfatide ratio for MS myelin. Analysis of the fatty acid pattern of phospholipids in MS brains revealed lower values for 18:1 and, except for the lyso compounds, slightly higher amounts for 20:4 and 22:6. Lower levels for the sum of 18:3 and 20:1 fatty acids were found for the EPG and IPG fractions.

The synthesis in vivo of choline and ethanolamine phosphoglycerides in different brain areas during aging.

The biosynthesis of choline and ethanolamine phosphoglycerides was tested in vivo in different brain areas of the rat during aging. Mixtures of [2−3H] glycerol and [Me-14C] choline or [2-3H] glycerol and [2-14H] ethanolamine were injected into lateral ventricle of the brain as lipid precursors and their incorporation into corresponding phospholipid was examined. A significant decrease of synthesis of both phosphoglycerides takes place in cerebral cortex and in the striatum, and is already apparent at 9 months of age with no further decrease or change therafter. No significant change takes place in the cerebellum. The unchanged absorption of injected water-soluble precursors, together with the lack of any significant change of phospholipid/protein ratio in all examined brain areas, suggests that the incorporation of both glycerol and nitrogen bases are affected by aging.

Biosynthesis of rat brain phosphatidylcholines from intracerebrally injected choline.

Abstract— [Me‐3H] Choline was injected intracerebrally into male rats and the brains immediately removed by particular procedures at regular intervals over the first 1200 s. The incorporation of radioactivity into brain phosphorylcholine, CDP‐choline and phosphatidylcholines was examined and quantitated, in order to investigate the relative roles of net synthesis and base‐exchange reactions for choline incorporation into lipid. The molecular subspecies of phosphatidylcholines were also examined after isotope administration. Phosphorylcholine, CDP‐choline and phosphatidylcholines all became labelled as early as 5 s after the administration of labelled choline. The time course of incorporation of choline into brain lipid is biphasic with two flex points at about 20 and 120 s from the injection. The specific radioactivity of different phosphatidylcholines appears to be different at early and later intervals from injection. The suggestion is made that the base‐exchange pathway for choline incorporation into lipid might be operative in vivo in early periods after administration.

Zur Aktivität der Phospholipase A2 gegenüber verschiedenen 1-Alk-1′-enyl-2-acyl-und 1-Alkyl-2-acyl-Verbindungen während der multiplen Sklerose

SummaryThe 1,2-diacyl- and 1-alk-1′-enyl-2-acyl portion of ethanolamine-phosphoglycerides was determined in white matter taken from brains of 9 patients with multiple sclerosis and from 9 control brains. MS brains showed a significant decrease of ethanolamine-plasamlogen, whereas the concentration of total ethanolamine-phosphoglycerides was not altered.2-acyl-1-alk-1′-enyl- and 2-acyl-1-alkyl-sn-glycerin-3-phosphorylcholin, specifically labelled at the 2-position with different fatty acids, were prepared enzymatically by using the acyltransferase system of rabbit sarcoplasmic reticulum. The substrates were subjected to hydrolysis by phospholipase A2 prepared from pathological (MS) and control brains. Alkyl- and alkenyl-derivatives, esterified with linoleic acid at the 2-position, were hydrolyzed at similar rates by the enzyme. The phospholipase A2 removed polyunsaturated fatty acids, such as linolenic and arachidonic acids, less actively from choline-plasmalogen. With the multiple sclerosis material an increase of approximately 50% in phospholipase A2 activity was observed in all substrates studied.ZusammenfassungBei jeweils 9 Multiple Sklerose- und Kontrollgehirnen wurde die Äthanolamin-Phosphatid-Fraktion der normalen weißen Substanz auf ihren Anteil an 1,2-Diacyl- und 1-Alk-1′-enyl-2-acyl-sn-glycerin-3-phosphoryläthanolamin untersucht. Die MS-Gehirne wiesen eine deutliche Verminderung des Äthanolamin-Plasmalogen-Anteiles auf; die Äthanolamin-Phosphatid-Fraktion war nicht vermindert.Mit Hilfe von Acyltransferase aus sarkoplasmatischem Reticulum des Kaninchens wurde auf enzymatischem Wege in der 2-Stellung mit verschiedenen Fettsäuren spezifisch markiertes 2-Acyl-1-alk-1′-enyl- und 2-Acyl-1-alkyl-sn-glycerin-3-phosphorylcholin dargestellt. Die Substrate wurden der Hydrolyse durch eine Phospholipase A2, welche aus MS- und Normalgehirnen dargestellt wurde, unterworfen. Die mit der Linolsäure veresterten Alkyl- und Alkenylderivate wurden von dem Enzym etwa gleich schnell gespalten. Die spezifische Aktivität der Phospholipase A2 nahm ab, sobald sich in der 2-Stellung der Cholin-Plasmalogene höher ungesättigte Fettsäuren wie die Linolen- oder Arachidonsäure befanden. Bei der multiplen Sklerose war die Aktivität der Phospholipase A2 gegenüber allen untersuchten Substraten signifikant um etwa 50% erhöht.

The metabolism of labelled choline in neuronal and glial cells of the rabbit in vivo.

Abstract— Adult rabbits were injected intraventricularly with [14C]ethanolamine and the incorporation of the base into the phosphatidylethanolamine and ethanolamine plasmalogen (and their water‐soluble precursors) of isolated neuronal and glial cells was investigated. All the radioactivity was incorporated into the base moiety of the ethanolamine lipids for the time intervals examined in both types of cells. In neurons, maximum labelling of the two ethanolamine lipids occurred at 7 h after administration, whereas the highest specific radioactivity for glial phosphatidylethanolamine and ethanolamine plasmalogen was reached at 20 and 36 h, respectively. The two lipids had a faster turnover in neurons than in glia, and in both populations incorporated the base at a faster rate than did whole brain tissue. The maximum incorporation rates for phosphorylethanolamine and CDP‐ethanolamine were reached in both types of cell at about 6 h after administration but the content of radioactivity per unit protein for phosphorylethanolamine was much higher in glial than in neuronal cells. It is concluded that the site of most active synthesis of ethanolamine phospholipids in vivo is the neuronal cell, with a possible transfer of intact lipid molecule to the glial compartment.

Glycerinphosphatide und Sphingolipide der normalen weißen Substanz bei der Multiplen Sklerose

SummaryConcentration and fatty acid composition of phospholipids and some sphingolipids were determined in normal white matter taken from brains of 11 patients with multiple sclerosis and 11 control brains by means of column, one- and two-dimensional thin-layer, and gas-liquid chromatography. In addition to total phospholipids we examined the choline-, ethanolamine- and serine-phosphogly-ceride fractions, phosphatidylinositol, monoacyl-sn-glycero-3-phosphorylcholine and-ethanolamine, sphingomyelin, cholesterol, cerebrosides and sulfatides. MS brains showed a significant decrease of the serine-phosphatide fraction whereas the lysophosphoglycerides were slightly elevated. Among the galactolipids sulfatides were found to be lowered; in comparison with the normal material the cerebroside/sulfatide ratio of MS brains was increased. Analysis of the fatty acid pattern of CGP, ÄGP, SGP, IGP and sphingomyelin revealed variously lowered values for oleic acid and elevated figures for the polyunsaturated long-chain fatty acids. ÄGP showed lower figures for the sum of 18:3 and 20:1. Lysophosphatides of MS brains showed an increase of the saturated fatty acids with a concomitant reduction of unsaturated fatty acids. The composition of unsubstituted fatty acids of sphingogalactolipids revealed higher values for 25:1 and the pattern of 2-hydroxy acids an increase of 24:1.ZusammenfassungBei jeweils 11 Multiple Sklerose- und Kontrollgehirnen wurde die normale weiße Substanz mit Hilfe der Säulen-, der ein- und zweidimensionalen Dünnschicht- und der Gaschromatographie auf Veränderungen im Gehalt und in der Fettsäurezusammensetzung der Phospholipide und einiger Sphingolipide untersucht. Außer den Gesamtphospholipiden analysierten wir im einzelnen die Cholin-, Äthanolamin- und die Serin-Phosphatid-Fraktion, Phosphatidylinositol, Monoacyl-sn-glycerin-3-phosphorylcholin und -äthanolamin, Sphingomelin, Cholesterin, die Cerebroside und die Sulfatide. Die MS-Gehirne zeigten einen deutlichen Abfall der Serin-Phosphatid-Fraktion und einen leichten Anstieg der Lysophosphatide. Die Galaktolipide wiesen eine Verminderung der Sulfatide und eine Erhöhung des Cerebrosid/Sulfatid-Verhältnisses bei den MS-Gehrinen im Vergleich zu den Normalgehirnen auf. Bei CGP, ÄGP, SGP, IGP und dem Sphingomyelin zeigte sich in unterschiedlichem Ausmaße eine Verminderung der Ölsäure und eine Zunahme der hochungesättigten langkettigen Fettsäuren. Die Summe von 18:3 und 20:1 war in der Äthanolamin-Phosphatid-Fraktion vermindert. Bei den Lysoverbindungen der MS-Gehirne wurde ein Anstieg der gesättigten und eine Verminderung der ungesättigten Fettsäuren beobachtet. Bei den Sphingogalaktolipiden fanden wir unter den unsubstituierten Fettsäuren eine Erhöhung von 25:1 und unter den 2-Hydroxyfettsäuren eine Zunahme von 24:1.

Relationships between base-exchange reaction and the microsomal phospholipid pool in the rat brain in vitro

The calcium-stimulated incorporation of ethanolamine, choline and L-serine into rat brain microsomal phospholipids has been investigated. The membranes were prelabeled in vitro in their choline or serine phosphoglycerides by base-exchange and then chasing experiments were done by displacing the lipid-bound base by ethanolamine, choline, or L-serine labeled with a different isotope. The results indicate that membrane phosphatidylcholine is presumably a substrate for the exchange with all the three bases, whereas phosphatidylserine exchanges only with ethanolamine and L-serine but not with choline. A small phospholipid pool (3–7% of the total available pool) is active in the calcium-dependent exchange with choline, ethanolamine, and L-serine. When the microsomal membranes are prelabeled in vitro in their phosphatidylcholine moiety through the cytidine-dependent pathway and then chasing experiments are performed with the three nitrogenous bases, as above, the small phospholipid pool is hardly detectable. In view of these and other results (Gaiti et al., FEBS Letters 49:361 1975), it is suggested that at least two different pools of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine might exist in rat brain microsomes.

MEMBRANE-BOUND BASE-EXCHANGE REACTIONS IN ANIMAL TISSUES

The calcium-stimulated incorporation of ethanolamine, L-serine and choline into rabbit synaptosomal phospholipids in vitro has been investigated. The synaptosomal membranes were prelabelled in vitro in their choline-, ethanolamine- or serine-phosphoglycerides by base-exchange and then chasing experiments were performed by displacing the lipid-bound base by ethanolamine, choline or L-serine labelled with a different isotope. The results indicate that membrane phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine are substrates for the exchange with all the three mentioned bases. A very small phospholipid pool (0.5-2% of the total available pool) is active in the calcium-dependent exchange between membrane phosphatidylcholine or phosphatidylethanolamine and free bases, whereas the pool of exchanging phosphatidylserine is sensibly larger (2-9%). In another series of experiments the effect of the base-exchange reaction upon the production of cyclic-AMP at the level of rat brain synaptic membranes has been examined. An exchange with ethanolamine produces a significant decrease of the NaF-stimulated production of the cyclic nucleotide, whereas it increases the noradrenaline-induced production. With some exceptions, the exchange with L-serine produces opposite effects. The possible physiological importance of phospholipid pool at the synaptosomal level is discussed.

Metabolic Studies on 1-Alkyl- and 1-Alk-1′-Enyl-Glycerophosphat Ides in the Brain

Marked differences in phospholipid metabolism have been observed between glial and neuronal cell enriched fractions. Recently, evidence was obtained for a faster tuin nover of glycerophospholipids in neurons than in glial cells, since neurons contained a considerably higher phospholipase A1 and A2 activity when compared to the glial cell enriched fraction (l). Furthermore the neuronal cell bodies were found to possess a much higher rate of exchange of both serine and ethanolamine into the phospholipids than the glial cell enriched fraction (2), and Freysz et al, (S) showed in their extensive in. vestigat ion on the kinetics of the biosynthesis of phos pholipids in neurons and glial cells, isolated from rat brain cortex, that neuronal phospholipids had a faster turnover than glial phospholipids. Recent results, presented by Woelk et al. (4), indicated that phosphatidyl inositol and phosphatidylcholine had the fastest and ethanolamine plasmalogen the slowest turnover in both cell types. Studies on ethanolamine phosphoglyceride synthesis in isolated neuronal and glial cells of the rabbit in vitro (5/6) Ied to the conclusion that the cytidine-dependent enzymic system for ethanolamine and choline phosphoglyceride synthesis is present in both cell populations, being concentrated mostly in the neuronal cells as compared to glia. Recent studies on the metabolism of labelled ethanolamine in neuronal and glial cells of the rabbit indicated that the site of most active synthesis of ethanolamine phospholipids in vivo is the neuronal cell, with a possible transfer of intact lipid molecule to the glial compartment (7).