THE METABOLISM OF LABELLED ETHANOLAMINE IN NEURONAL AND GLIAL CELLS OF THE RABBIT IN VIVO1

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.     

ANALYSIS OF THE MAJOR AMINO ACIDS OF RAT BRAIN AFTER IN VIVO INHIBITION OF GABA TRANSAMINASE BY ETHANOLAMINE O-SULPHATE

The effect of in vivo inhibition of GABA transaminase by ethanolamine O-sulphate on the content of the free amino acids in rat brain has been studied. Intracisternal injection of 2.0 mg/kg resulted in a progressive increase in GABA levels with time, to reach after 8 h a 100 per cent increase over saline-injected control animals. The effect of injection of 0.5, 1.0 and 2.0 mg/kg was studied 24 h after injection and the results showed that the increased GABA levels were dependent on the dose of inhibitor employed. Apart from the substantial increase in the GABA concentration of the brain there were no significant changes in the content of the other amino acids except for a small but significant decrease in aspartic acid in one experiment. When the extent of inhibition of the transaminase was correlated with the rise in GABA concentration it was shown that no elevation occurred until more than half of the enzymic activity had been inhibited.     

THE INFLUENCE OF PORTOCAVAL ANASTOMOSIS ON THE METABOLISM OF LABELLED OCTANOATE, BUTYRATE AND LEUCINE IN RAT BRAIN

Rats were given a portocaval anastomosis and 3 weeks later, when the only ultrastructural change in the CNS is watery swelling of astrocytes, several aspects of brain metabolism were studied. The uptake of leucine by the brain, its incorporation into protein and its oxidation were followed after the simultaneous injection of a mixture of L-[1¹⁴C]leucine and L-[4,5-³H]leucine. The concentration of leucine in blood was lowered in the operated animals whereas in brain it was increased. The specific radioactivity of leucine in the brain was comparable to values in control animals and there was no evidence of a decrease in incorporation of [1-¹⁴C]leucine into brain proteins over the short experimental time period studied. The only difference from the controls in the oxidation of [4,5-³H]leucine was a greater accumulation in glutamine. The amount of glutamine in the brains of the operated animals had increased 4-fold at the time of the metabolic studies. From dual-labelled experiments in which a mixture containing [1-¹⁴C]butyrate and L-[4,5-³H]leucine was injected intravenously, it was shown that, in both control and operated animals, the pools of brain glutamate and glutamine labelled from butyrate were metabolically distinct from those labelled from leucine. The total radioactivity appearing in brain from [1-¹⁴C]butyrate was markedly reduced in operated animals, but the radioactivity from L-[4,5-³H]leucine was not. The metabolism of [1-¹⁴C]octanoate was compared with that of [1-¹⁴C]butyrate. In control animals the labelling of metabolites was almost identical with either precursor. In operated animals there was no reduction in the uptake of [1-¹⁴C]octanoate into the brain. There was evidence that the size of the glutamine pool labelled, relative to glutamate, was increased but that it had a slower fractional turnover coefficient. A link between astroglial changes and an impairment to the carrier mechanism for transport of short chain monocarboxylic acids across the blood-brain barrier is suggested.     

PROPERTIES AND METABOLISM OF SOLUBLE LIPOPROTEINS CONTAINING CHOLINE AND ETHANOLAMINE PHOSPHOLIPIDS IN RAT BRAIN

Abstract— Choline and ethanolamine phospholipids in the 105,000 g supernatant fraction of rat brain exhibited density and electrophoretic properties consistent with their binding to protein. About 40% of these two phospholipids were bound to soluble lipoproteins, whereas the remainder appeared to be associated with particulate complexes. Following intracranial injection of [2-³H]glycerol, the specific radioactivities of the choline and ethanolamine phospholipids in the supernatant fraction were higher than those in the microsomal fraction at all time points examined, from 15 min to 12 h after injection. The properties of cytoplasmic lipoproteins containing choline and ethanolamine phospholipids have been compared with those which we have previously described containing sulphatide.     

METABOLISM IN VIVO OF BRAIN GALACTOLIPIDS: THE JIMPY MUTANT

Abstract— The incorporation in vivo of [U-¹⁴C]glucose into the galactolipids of the brain of control and Jimpy mutant mice was examined. Over a 24-h period of incorporation there was no indication of an increased rate of turnover of brain galactolipids in the mutant. The Jimpy mutant was identified at ages prior to and at the inception of myelination (7–10 days post partum) with a coat marker (Tabby). There was similar total radioactivity in galactolipids of the Jimpy at these ages but a reduction to 13 per Cent of control at 13 days and to 6 per cent at 16 days of postnatal age. This devetopmental pattern of galactolipid synthesis in Jimpy brain is not in accord with a primary defect in the biosynthesis of cerebrosides and sulphatides.     

EFFECTS OF LITHIUM TREATMENT IN VITRO AND IN VIVO ON ACETYLCHOLINE METABOLISM IN RAT BRAIN

Abstract— The effects of LiCl on cholinergic function in rat brain in vitro and in vivo have been investigated. The high affinity transport of choline and the synthesis of acetylcholine in synaptosomes were reduced when part (25-75%) of the NaCl in the buffer was replaced with LiCl or sucrose. This appeared to be due to lack of Na⁺ rather than to Li⁺, as addition of LiCl to normal buffer had little effect. Following an injection of LiCl (10mmol/kg, i.p.) into rats the concentration of a pulsed dose of [²H₄]choline (20 μmol/kg, i.v., 1 min) and its conversion to [²H₄]acetylcholine, and the concentrations of [²H₂]acetylcholine and [²H₀]choline were measured in the striatum, cortex, hippocampus and cerebellum. The [²H₄]choline and [²H₄]acetylcholine were initially (15 min after LiCl) reduced (to ?30% in the cortex) and later (24 h after LiCl) increased (to + 50% in the striatum). There was a corresponding initial increase (to +50% in the cerebellum) and later decrease (to ?30% in the hippocampus) of the endogenous acetylcholine and choline. These results indicate an initial decrease and later increase in the utilization of acetylcholine after acute treatment with LiCl. Following 10 days of treatment with LiCl there was an increased rate of synthesis of [²H₄]acetylcholine from pulsed [²H₄]choline in the striatum, hippocampus and cortex (P < 0.05). The high affinity transport of [²H₄]choline and its conversion to [²H₄]acetylcholine was activated (131% of control; P < 0.01) in synaptosomes isolated from brains of 10-day treated rats. Investigation of synaptosomes isolated from striatum, hippocampus and cortex revealed that only striatal [²H₄]acetylcholine synthesis was significantly stimulated. Kinetic analysis demonstrated that the apparent K_T for choline was decreased by 30% in striatal synaptosomes isolated from rats treated for 10 days with LiCl. Striatal synaptosomes from 10-day treated rats compared to striatal synaptosomes from untreated rats also released acetylcholine at a stimulated rate in a medium containing 35 mM-KCl. These results indicate that LiCl treatment stimulates cholinergic activity in certain brain regions and this may play a significant role in the therapeutic effect of LiCl in neuropsychiatric disorders.     

METABOLISM OF THE ASPARTYL MOIETY OF N-ACETYL-l-ASPARTIC ACID IN THE RAT BRAIN

The metabolism of N-acetyl-l -aspartic acid (NAA) was studied in rat brain. [Aspartyl-U-¹⁴C]NAA was metabolized predominantly by deacylation. Studies of NAA biosynthesis from l -[U-¹⁴C]aspartic acid have confirmed previous reports that NAA turns over slowly in rat brain. However, intracerebrally-injected N-acetyl-l -[U-¹⁴C]asparticacid was rapidly metabolized. Exogenous NAA appears to be taken up rapidly into a small, metabolically-active pool. This pool serves as substrate for a tricarboxylic acid cycle associated with the production of glutamate for the biosynthesis of glutamine. The bulk of the NAA content in brain appears to be relatively inactive metabolically.     

THE UPTAKE OF PURINES BY RAT BRAIN IN VIVO AND IN VITRO

Abstract— The uptake of [¹⁴C]guanine and some of its [¹⁴C]-labelled derivatives into rat brain was studied in vivo and in vitro. In vivo guanine, guanosine, and hypoxanthine penetrated the brain of adult rats to a very small extent. Inosine was taken up somewhat better. In young animals, also, guanosine was taken up poorly, but guanine was taken up fairly well. When guanine was administered to adult animals, only guanine was found in the brain. In young animals, by contrast, radioactivity from guanine appeared in guanosine and in guanine nucleotides, but no free guanine was found. In vitro guanine was taken up much better and, in fact, remained mostly as guanine in slices from 10-day-old rats. The in vitro conversion of guanine to GMP and its incorporation into RNA was unimpaired by the addition of unlabelled guanosine, an indication that guanine was converted directly to GMP. The uptake of guanine in vitro was not subject to competitive inhibition or influenced by the presence of dinitrophenol. This finding suggested that guanine entered the slice by simple diffusion.     

IN VIVO METABOLISM OF [G-3H]LIGNOCERIC ACID IN DEVELOPING RAT BRAIN1

Abstract— [G-³H]Lignoceric acid (tetracosanoic acid) was injected into the brains of 20-day-old rats, and the animals were killed after 8, 24, or 72 h. Various lipids were isolated from these brains, and the distribution of radioactivity was determined. The injected free acid rapidly disappeared, and the radioactivity was incorporated into varying chain-length nonhydroxy- and hydroxy saturated fatty acids of sphingolipids and phospholipids. Little radioactivity was found in unsaturated acids, sphingo-sine, and cholesterol. A time-dependent shift of the label among various fatty acids was relatively small 8 h after injection, probably because of the metabolic stability of the brain sphingolipids. In cerebrosides, the radioactivity was equally distributed between nonhydroxy and x-hydroxy fatty acids of all chain lengths. C₂₃ and C₂₂ fatty acids contained equal total radioactivities; C₂₃ and C₂₄ fatty acids contained similar specific activities. These results confirm the significant role of a-hydroxylation and 2-oxidation in the synthesis of very long-chain fatty acids in brain. In total lipid fatty acids, docosanoic acid (22:0) contained more radioactivity than its α-oxidation precursor, α-hydroxytricosanoic acid (23h:0) at all times. In sphingolipid fatty acids, the specific activity of 21:0 was always higher than that of its ct-oxidation precursor 22:0. These observations indicate that part of the 22:0 and 21:0 was derived by β-oxidation from the injected lignoceric acid or its α-oxidation product, respectively.     

MEASUREMENT OF THE RATE OF GLUCOSE UTILIZATION BY RAT BRAIN IN VIVO

Abstract— A method is described by which the rate of glucose utilization by whole brain of conscious rats may be measured. The basis is the uptake of ¹⁴C derived front [2-¹⁴C] glucose into the acid-soluble metabolite pool of brain. Catheters are placed in the femoral artery and vein under light ether anesthesia. After full recovery of consciousness a single intravenous injection of [2-¹⁴C] glucose is given and arterial blood samples taken at intervals. Simultaneous with the last sample the brain is removed and frozen within 1 s. The accumulation of ¹⁴C into the acid-soluble metabilite pool is measured and the rate of glucose utilization is calculated according to the equation:

The integral is calculated from the plasma glucose specific activity curve and evidence is presented to justify this procedure. The rate of glucose utilization measured by this method was 0·62 μmol/min per g in conscious rats and 0·28 μmol/min per g in sodium pentobarbital anesthetized rats.     

NUCLEOTIDE METABOLISM IN RAT BRAIN

Abstract— The uptake, the conversion to nucleotides, and their incorporation into RNA for labelled glycine, aspartate, the free bases and nucleosides of purines and pyrimidines were investigated with cortical slices of rat cerebrum. At the end of a 1-hr incubation time the slice-to-medium ratio of the radioactivities for labelled aspartate, glycine, adenine and adenosine were 34, 26, 20 and 5, respectively, while the slice-to-medium ratios for hypoxanthine, inosine, guanine, guanosine, xanthine, orotate, cytidine, cytosine, uridine, and uracil ranged from 1.3:1 to 2:1. Over 99 per cent of the total radioactivity taken up by the cortical slices was present in the TCA supernatant and 86, 82, 65, 50, 34, 23, 20 and 1.6 per cent of this radioactivity was in the form of nucleotides at the end of a 1-hr incubation with labelled adenine, adenosine, hypoxanthine, inosine, uridine, orotate, cytidine, and glycine, respectively. The incorporation of various radioactive precursors into RNA of cortical slices suggests that nucleotides originating from either de novo synthesis or preformed purine derivatives enter the same nucleotide pool utilized for RNA synthesis. The supernatant fraction from homogenized cerebrum was investigated for the presence of various anabolic and catabolic enzymes associated with nucleotide metabolism. These results were correlated with the data from the RNA incorporation studies, and a possible role for AMP: pyrophosphate phosphoribosyltransferase (adenine phosphoribosyltransferase, I.U.B. 2.4.2.7) to achieve intercellular transfer of AMP is discussed.     

DEVELOPMENT OF MITOCHONDRIAL PYRUVATE METABOLISM IN RAT BRAIN

The activities of a number of mitochondrial enzymes involved in the metabolism of pyruvate during development of the rat brain were investigated. The rates of decarboxylation of [1-¹⁴C]pyruvate to ¹⁴CO₂ via pyruvate dehydrogenase and the fixation of H¹⁴CO₃^? in the presence of pyruvate via pyruvate carboxylase by brain homogenates were very low in newborn rats. These rates increased markedly by about four-fold and 15-fold respectively during 10–35 postnatal days. The rates of the fixation of H¹⁴CO₃^? by cerebral homogenates were supported by the development of the activity of pyruvate carboxylase in rat brain. The activities of citrate synthase, aconitase, NAD-malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and phosphoenol-pyruvate carboxykinase were very low in the particulate fraction of the newborn rat brain. The activities of all these enzymes increased makedly by about three- to 10-fold during 10–35 days after birth. The activity of mitochondrial phosphoenolpyruvate carboxykinase from rat brain was not precipitated by an antibody prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase suggesting that cerebral mitochondrial enzyme is immunologically different from that of the cytosolic form in hepatocytes. The significance of the development of the cerebral mitochondrial metabolism is discussed in relation to biochemical maturation of the brain.     

SYNTHESIS AND METABOLISM OF l-KYNURENINE IN RAT BRAIN

Abstract— A method for the quantitative analysis of femtomole amounts of kynurenine (along with tryptophan, 3-hydroxykynurenine and kynuramine) in rat brain using high pressure liquid chroma-tography and electron-capture GLC is described. Endogenous concentrations of these substances in rat brain regions were measured, and their formation after the injection of radioactive tryptophan or kynurenine was determined. Kynurenine was formed from tryptophan in brain and was also taken up from the periphery. Extracerebral kynurenine was calculated to account for 60% of the cerebral pool of kynurenine. The cerebral rates of synthesis of kynurenine and 3-hydroxykynurenine were 0.29 and 0.17nmol/g/h. The turnover rate of kynurenine in the brain was 1.02 nmol/g/h measured from [¹⁴C]tryptophan or 1.14 nmol/g/h from [³H]kynurenine injected intraperitoneally. Kynuramine levels in different areas of the brain were similar to those of tryptamine. Following intraperitoneal injection of [¹⁴C]tryptophan, the presence of anthranilic, 3-hydroxyanthranilic, xanthurenic, kynurenic and quinaldic acids was demonstrated in the brain.     

THE TURNOVER OF ISOTOPICALLY LABELLED DNA IN VIVO IN DEVELOPING, ADULT AND SCRAPIE-AFFECTED MOUSE BRAIN

Abstract— Tracer experiments using [³H]thymidine have shown that a large proportion of the DNA synthesized in control and scrapie-affected mouse brain is metabolically unstable. Although the turnover of mitochondrial DNA contributed to the loss of radioactivity from whole brain, it has been shown that 70 per cent of the labelled nuclear DNA was removed between 1 and 21 days after injecting the isotopic precursor. Observations on developing mouse brain, where the rate of DNA synthesis is far higher than that in adult brain, also revealed the existence of metabolically unstable DNA. Similar studies on developing and adult brain using [¹⁴C]thymidine indicated that most of the radioactivity lost in vivo was not due to radiation damage to newly labelled DNA molecules. Hydroxyapatite chromatography of heat denatured and renatured DNA from adult brain showed that the rates of turnover of the poorly, moderately and highly reiterated species of nuclear DNA were similar. The results of some dissection experiments have further shown that the observed breakdown of DNA in adult brain was not specifically associated with the turnover of subependymal cells. It is suggested that a metabolically labile fraction of nuclear DNA is present in developing and adult mouse brain and that the amount of tracer incorporated into this fraction is increased in mice infected with scrapie.     

OBSERVATIONS ON THE BIOSYNTHESIS OF RAT BRAIN CERAMIDE AND CEREBROSIDE IN VIVO

Abstract— The incorporation in vivo of l -[¹⁴C]serine into ceramide and cerebroside of young rat brain has been studied. Acid hydrolysis of labelled ceramide and galactosyl-ceramide followed by selective partitioning of the resulting components indicated that 88 per cent of the radioactivity was present in the long-chain base portion. At early time points (10 min, 20 min) the precursor was incorporated into ceramide and to a lesser degree into glucosyl-ceramide. During time intervals of 5 and 10 h, the specific activity values (d.p.m./μmol) for ceramide and glucosyl-ceramide decreased, while values for galactosyl-ceramide, containing either unsubstituted fatty acids (NFA) or α-hydroxy fatty acids (HFA), increased 50 and 30 per cent, respectively. Analysis of labelled ceramide at all time points studied (10 min-10 h) indicated that l -[¹⁴C]serine was incorporated onto the NFA type. This observation suggests that HFA-ceramide may not be the physiological precursor of HFA-galactosyl-ceramide. In this context, the postulated precursor roles of both ceramide and psychosine in the biosynthesis of brain cerebrosides are discussed.     

THE METABOLISM OF TRITIATED DOPAMINE IN REGIONS OF THE RAT BRAIN IN VIVO—I

Abstract— A simple combination of acid- and base-exchange resin columns enabled a more complete separation of amine, acid and neutral metabolites of catecholamines from individual small brain samples to be made. Fractions containing individual catecholamines or metabolites were obtained in aqueous eluates suitable for fluorimetric or radioisotopic analysis. With consistent intraventricular injection and brain dissection techniques, this separation method enables a study of the metabolism of catecholamines in regions of the rat brain and the effects of drugs on this metabolism.     

THE METABOLISM OF TRITIATED DOPAMINE IN REGIONS OF THE RAT BRAIN IN VIVO—II

Abstract— The levels of tritiated catecholamines and metabolites were measured in regions of the rat brain at intervals after the intraventricular injection of [³H]dopamine, [³H]nor-adrenaline and [³H]normetanephrine. The disappearance of catecholamines and appearance of metabolites with time and the regional turnover rates of these amines indicate that the major pathway of the metabolism of noradrenaline and dopamine actively released from physiological storage sites is to the neutral alcoholic metabolites. The acid metabolites, homovanillic acid and 3,4-dihydroxyphenylacetic acid appear to be only minor products of normal dopamine metabolism in rat brain regions including the striate, but are the main end products of the metabolism of excess exogenous dopamine.
The active metabolism of stored noradrenaline to alcohol metabolites is also indicated by the increase in neutral alcohol metabolites accompanying the increased noradrenaline turnover when rats were subjected to electroshock stress. Therefore in the rat brain, neutral alcohol metabolites of dopamine and noradrenaline have great significance in the study of physiological catecholamine turnover in any region.     

IN VIVO METHYLATION AND TURNOVER OF RAT BRAIN HISTONES

Abstract— The turnover of the different histone components from brain nuclei was studied after the administration of l -[³H]lysine and l -[¹⁴C-methyl]methionine to newborn rats. The radioactivities of the different histone subfractions as well as other proteins were determined over a 280-day period. Biphasic type decay curves (³H and ¹⁴C) were obtained for total brain histones and all the subfractions. From 6 to 40 days of age the half life of total brain histones was 19 days. After reaching brain maturity the half life was 132 days. The lysine rich histone (F₁) was found to turnover the fastest of all the histones, having half lives of 13 and 112 days, respectively. The decay curve for the slightly lysine rich histones (F_2a2, F_2b) gave half lives of 25 days up to 40 days of age and 189 days after reaching brain maturity. The arginine rich histones (F_2a1, F₃) gave a half life of 32 days up to 40 days of age, while no turnover was observed after maturity. The turnover rates of the methyl groups and/or methionyl residues did not vary significantly from the turnover rates of the lysyl residues in the F₂ and F₃ histones. The lysine-rich histones did not contain significant amounts of methionyl residues or methyl groups.
Amino acid analysis of the brain histones revealed that about 3·6 per cent of the lysyl residues in the slightly lysine rich histones were methylated, mainly as ε-N-dimethyllysine. About 13 per cent of the lysyl residues in the arginine rich histones were methylated, mainly as ε-N-monomethyllysine and ε-N-dimethyllysine.     

REGIONAL BRAIN STUDY OF INDOLEAMINE METABOLISM IN THE RAT IN ACUTE HEPATIC FAILURE

Abstract— Tryptophan, 5-hydroxytryptamine and 5-hydroindoleacetic acid were found to be greatly increased in various parts of the brains of rats in acute hepatic failure following two stage hepatic devascularization. However, the increases in 5-hydroxytryptamine and 5-hydroxyindoleacetic acid varied by region and are not explicable solely in terms of increased concentrations of tryptophan. The results are discussed in terms of differences in the regional metabolism of 5-hydroxyindoleamines. Plasma free fatty acids, albumin, total tryptophan and free tryptophan were measured in plasma in hopes of elucidating the mechanism responsible for the cerebral elevation of tryptophan. Increased plasma free tryptophan appears sufficient to explain the rapid increase in brain tryptophan. The relationship between these results and recent observations in hepatic encephalopathy is discussed.