Experiments in marine biochemistry. Homarine metabolism in Penaeus duorarum.

A fractionation procedure has been developed which permits the isolation of 1 to 2 mg of homarine from a single shrimp. This procedure was used to show that homarine is endogenously synthesized by Penaeus duorarum in the free unbound form, and to study the metabolic precursors involved. Injected DL-[14C]tryptophan was not converted to [14C]homarine. However, [6-14C]quinolinic acid, a known catabolite of tryptophan, is an effective precursor. [2-14C]Acetate and [U-14C]glycerol are effectively converted to [14C]homarine while [14C]bicarbonate is poorly utilized. The injection of L-[U-14C]aspartate resulted in labeled homarine, but the quantity converted was less than expected. Since [14C]glycerol is an effective precursor there is a possibility that quinolinic acid may be formed in P. duorarum by a condensation similar to that of glyceraldehyde 3-phosphate with aspartic acid or a closely related metabolite. It is suggested that decarboxylation of quinolinic acid gives rise to picolinic acid which is methylated to yield homarine. L-[methyl-14C]Methionine efficiently provides the N-methyl carbon presumably via S-adenosylmethionine.     

The metabolism of L-tryptophan by isolated rat liver cells. Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism.

1. The metabolism of L-tryptophan by liver cells prepared from fed and 48 h-starved rats was studied. Methods are described, with the use of L-[ring-2-(14)C], L-[carboxy-14C]-and L-[benzene-ring-U-14C]-tryptophan, for the simultaneous determination of tryptophan 2,3-dioxygenase and kynureninase activities and of the oxidation of tryptophan to CO2 and non-aromatic intermediates of the kynurenine-glutarate pathway. 2. At physiological concentrations (0.1 mM), tryptophan was oxidized by tryptophan 2,3-dioxygenase at comparable rates in liver cells from both fed and starved rats. Kynureninase activity of hepatocytes from starved rats was 50% greater than that of cells from fed rats. About 10% of the tryptophan metabolized by tryptophan 2,3-dioxygenase was degraded completely to CO2. 3. In the presence of 0.5 mM-L-tryptophan, tryptophan 2,3-dioxygenase and kynureninase activities increased 5--6-fold. Liver cells from starved rats oxidized tryptophan at about twice the rate of these from fed rats. Degradation of tryptophan to non-aromatic intermediates of the glutarate pathway and CO2 was increased only 3-fold, suggesting an accumulation of aromatic intermediates of the kynurenine pathway. 4. Rates of metabolism with 2.5 mM-L-tryptophan were not significantly different from those obtained with 0.5 mM-tryptophan. 5. Rates of synthesis of quinolinic acid from 0.5 mM-L-tryptophan, determined either by direct quantification or indirectly from rates of radioisotope release from L-[carboxy-(14)C]- and [benzene-ring-U-14C]tryptophan, were essentially similar. 6. At all three concentrations examined, tryptophan was degraded exclusively through kynurenine; there was no evidence of formation of either indol-3-ylacetic acid or 5-hydroxyindol-3-ylacetic acid.     

MEDIATION BY β-ADRENERGIC RECEPTORS OF EFFECT OF NOREPINEPHRINE ON PINEAL SYNTHESIS OF [14C]SEROTONIN AND [14C]MELATONIN

Rat pineal organs maintained in organ culture converted [¹⁴C]tryptophan to [¹⁴C]serotonin and [¹⁴C]melatonin. The synthesis of both indoles was stimulated by the presence of norepinephrine or dibutyryl adenosine 3′,5′-monophosphate. This effect of norepinephrine could be blocked by the α-adrenergic blocking drug, propranolol, but was not modified by the a-adrenergic blocking agent, phenoxybenzamine. Neither blocking agent modified the pineal response to dibutyryl adenosine 3′,5′-monophosphate. Unlike dibutyryl adenosine 3′,5′-monophosphate, the naturally occurring adenosine phosphates did not stimulate synthesis of [¹⁴C]melatonin in vitro.     

Studies on the transport of acetyl groups from peroxisomes to mitochondria in isolated liver cells oxidizing the polyunsaturated fatty acid 22:4n-6.

The oxidation of the fatty acid [1-(14)C]22:4n-6 was studied in isolated hepatocytes. Labeled acetate was the main acid soluble product identified by HPLC after short incubation periods. At low substrate concentrations and longer incubations [(14)C]acetate was gradually replaced by labeled beta-hydroxybutyrate, acetoacetate and oxaloacetate/malate. Preincubation with 2-tetradecylglycidic acid (TDGA), an inhibitor of mitochondrial fatty acid oxidation, did not reduce the oxidation but acetate was the only product recovered. TDGA also strongly inhibited the metabolism of added [1-(14)C]acetate to mitochondrial oxidation products. During the preparation procedure of hepatocytes the cellular L-carnitine concentration was decreased but it was restored after preincubation with L-carnitine. With low [1-(14)C]22:4n-6, concentrating a low level of [(14)C]acetate and high levels of labeled mitochondrial oxidation products were recovered after preincubation with L-carnitine. A small amount of [(14)C]acetylcarnitine was also detected under this incubation condition. The results suggest that a significant part of labeled acetyl groups from the peroxisomal oxidation of [1-(14)C]22:4n-6 is transported to the mitochondria as free acetate. Moreover, the results also suggest that L-carnitine at physiological concentrations may facilitate the transport of part of the acetyl groups from peroxisomes to mitochondria as acetylcarnitine. However, the possibility that an increased cellular L-carnitine concentration may stimulate oxidation of [1-(14)C]22:4n-6 in mitochondria could not be excluded.     

Metabolic Stability of 3-O-Methyl-d-Glucose in Brain and Other Tissues

3-O-Methyl-D-glucose (methylglucose) is often used to study blood-brain barrier transport and the distribution spaces of hexoses in brain. A critical requirement of this application is that it not be chemically converted in the tissues. Recent reports of phosphorylation of methylglucose by yeast and heart hexokinase have raised questions about its metabolic stability in brain. Therefore, we have re-examined this question by studying the metabolism of methylglucose by yeast hexokinase and rat brain homogenates in vitro and rat brain, heart, and liver in vivo. Commercial preparations of yeast hexokinase did convert methylglucose to acidic products, but only when the enzyme was present in very large amounts. Methylglucose was not phosphorylated by brain homogenates under conditions that converted 97% of [U-14C]glucose to ionic derivatives. When [14C]methylglucose, labeled in either the methyl or glucose moiety, was administered to rats by an intravenous pulse or a programmed infusion that maintained the arterial concentration constant and total 14C was extracted from the tissues 60 min later, 97-100% of the 14C in brain, greater than 99% of the 14C in plasma, and greater than 90% of that in heart and liver were recovered as unmetabolized [14C]methylglucose. Small amounts of 14C in brain (1-3%), heart (3-6%), and liver (4-7%) were recovered in acidic products. Plasma glucose levels ranging from hypoglycemia to hyperglycemia had little influence on the degree of this conversion. The distribution spaces for methylglucose were found to be 0.52 in brain and heart and 0.75 in liver.     

Schistosoma mansoni: effects on tryptophan metabolism in mice

In mice, infection with 20-30 cercariae of Schistosoma mansoni resulted in a considerable reduction in the formation of 14CO2 from [14C]tryptophan. Infected animals excreted significantly lower amounts of kynurenine, kynurenic acid, and methyl pyridone carboxamide than did uninfected controls. There was no difference in the ability of hepatocytes isolated from infected or control animals to metabolise [14C]tryptophan. Hepatocytes from infected animals synthesized less NAD(P), but more niacin and N1-methyl nicotinamide from tryptophan. They showed no greater accumulation of kynurenine metabolites than did cells from control animals. The hepatocyte content of pyridoxal phosphate and the erythrocyte aspartate aminotransferase activation coefficient were the same in both groups of mice, suggesting that infection with S. mansoni does not deplete vitamin B6. The impairment of tryptophan metabolism in vivo was apparently not due to impaired hepatic metabolism. Rather, it seems likely that the parasites or their eggs take up tryptophan avidly from the host's circulation. Studies of parasite and egg metabolism of tryptophan may suggest novel approaches to the chemotherapy of bilharzia.     

Over-estimation of glucose-6-phosphatase activity in brain in vivo. Apparent difference in rates of [2-3H]glucose and [U-14C]glucose utilization is due to contamination of precursor pool with 14C-labeled products and incomplete recovery of 14C-labeled metabolites

Significant dephosphorylation of glucose 6-phosphate due to glucose-6-phosphatase activity in rat brain in vivo was recently reported (Huang, M., and Veech, R.L. (1982) J. Biol. Chem. 257, 11358-11363). The evidence was an apparent more rapid 3H than 14C loss from the glucose pool and faster [2-3H]glucose than [U-14C]glucose utilization following pulse labeling of the brain with [2-3H,U-14C]glucose. Radiochemical purity of the glucose and quantitative recovery of the labeled products of glucose metabolism isolated from the brain were obviously essential requirements of their study, but no evidence for purity and recovery was provided. When we repeated these experiments with the described isolation procedures, we replicated the results, but found that: 1) the precursor glucose pool contained detritiated, 14C-labeled contaminants arising from glucose metabolism, particularly 2-pyrrolidone-5-carboxylic acid derived from [14C]glutamine; 2) [14C]glucose metabolite were not quantitatively recovered; 3) the procedure used to isolate the glucose itself produced detritiated, 14C-labeled derivatives of [2-3H,U-14C]glucose. These deficiencies in the isolation procedures could fully account for the observations that were interpreted as evidence of significant glucose 6-phosphate dephosphorylation by glucose-6-phosphatase activity. When glucose was isolated by more rigorous procedures and its purity verified in the present studies, no evidence for such activity in rat brain was found.     

Serotonin synthesis and its light-dark variation in the rat retina

Retinal circadian rhythms are driven by an intrinsic oscillator, using chemical signals such as melatonin, secreted by photoreceptor cells. The purpose of the present work was to identify the origin of serotonin, the precursor of melatonin, in the retina of adult rat, where no immunoreactivity for serotonin or tryptophan hydroxylase had ever been detected. To demonstrate local synthesis of serotonin in the rat retina, substrates of tryptophan hydroxylase, the first limiting enzyme in the serotonin pathway, have been used. Tryptophan, in the presence of an inhibitor of aromatic amino acid decarboxylase, enhanced 5-hydroxytryptophan levels, whereas alpha-methyltryptophan, a competitive substrate inhibitor, was hydroxylated into alpha-methyl-5-hydroxytryptophan. Tryptophan hydroxylase substrate concentration was higher in the dark period than in the light period, and formation of hydroxylated compounds was increased. The presence of tryptophan hydroxylase mRNA in the rat retina was confirmed by RT-PCR. Taken together, the results support the local synthesis of serotonin by tryptophan hydroxylation, this metabolic pathway being required more critically when 5-HT is used for melatonin synthesis.     

Melatonin: A potential regulator of plant growth and development?

Summary Recent research has reported the presence of melatonin (N-acetyl-5-methoxytryptamine), a mammalian indoleamine neurohormone, in higher plants, indicating that melatonin may be an important metabolic regulator that has been highly conserved across biological kingdoms. Melatonin is synthesized from tryptophan in the mammalian pineal gland and a similar biosynthetic pathway was recently described in St. John's wort shoot tissues, wherein radiolabel from tryptophan was recovered in serotonin and melatonin as well as indoleacetic acid. There is growing information describing melatonin control of physiological processes in mammals, yeast, and bacteria, including diurnal responses, detoxification of free radicals, and environmental adaptations. However, at the current time, there is no known specific role for melatonin in plant physiology. Alterations in melatonin concentrations in plant tissues have been shown to affect root development, mitosis, and mitotic spindle formation. The recent advancements in melatonin research in plants and some directions for important areas of future research are reviewed in this article.     

Light emission from tryptophan oxidation by hypobromous acid

The emission of ultraweak light from cells is a phenomenon associated with the oxidation of biomolecules by reactive oxygen species. The indole moiety present in tryptophan, serotonin and melatonin is frequently associated with the emission of light during the oxidation of these metabolites. This study presents results for hypobromous acid (HOBr) oxidation of tryptophan as a putative endogenous source of ultraweak light emission. We found that chemiluminescence elicited by the oxidation of tryptophan by HOBr was significantly higher than by hypochlorous acid (HOCl). This difference was related to secondary oxidation reactions, which were more intense using HOBr. The products identified during oxidation by HOCl, but depleted by using HOBr, were N‐formylkynurenine, kynurenine, 1,2,3,3a,8,8a‐hexahydro‐3a‐hydroxypyrrolo[2,3‐b]‐indole‐2‐carboxylic acid, oxindolylalanine and dioxindolylalanine. The emission of light is dependent on the free α‐amino group of tryptophan, and hence, the indole of serotonin and melatonin, although efficiently oxidized, did not produce chemiluminescence. The emission of light was even greater using taurine monobromamine and dibromamine as the oxidant compared to HOBr. A mechanism based on bromine radical intermediates is suggested for the higher efficiency in light emission. Altogether, the experimental evidence described in the present study indicates that the oxidation of free tryptophan or tryptophan residues in proteins is an important source of ultraweak cellular emission of light. This light emission is increased in the presence of taurine, an amino acid present in large amounts in leukocytes, where this putative source of ultraweak light emission is even more relevant. Copyright © 2012 John Wiley & Sons, Ltd.     

Utilization of pyruvate and pyruvate precursors by normal and carcinogen-altered rat tracheal epithelial cells in culture

The metabolism of [14C]pyruvate, [14C]glucose, [14C]glutamine and [14C]alanine was compared between normal rat tracheal epithelial cells and carcinogen-altered cells derived from dimethylbenz(a)anthracene-exposed tracheal implants. Normal primary cultures (NPC) of tracheal cells are distinguished by their need for pyruvate-supplemented medium for growth and survival. The altered cells were selected out by their survival in the unsupplemented medium. Compared to the selected primary cultures (SPC), the NPC showed a three- to four-fold higher incorporation of radioactivity from [2-14C]pyruvate in all the macromolecular fractions, as well as in all the metabolites isolated from the acid soluble fraction and from lactic acid isolated from the medium. [U-14C]glucose was also incorporated at higher levels into lactic acid isolated from the acid soluble fraction and the medium of NPC. These data indicate a higher rate of glycolysis in the normal tracheal cells. This was supported by the findings of a two-fold greater glucose consumption and two-fold higher production of lactic acid isolated from the NPC medium. Lactate dehydrogenase activity was also two-fold higher in NPC. Thus, despite the apparently higher level of pyruvate production in the NPC, exogenous pyruvate is necessary to satisfy the metabolic needs of NPC. The utilization of [U-14C]glutamine or [U-14C]alanine was not markedly different between NPC and SPC. Furthermore, radioactivity from both of the amino acids was recovered in lactic acid in the medium, indicating that both cell types can derive pyruvic acid from either glutamine or alanine. SPC apparently do not use these routes to supply higher levels of pyruvic acid for survival in culture. The oxidation of none of the radioactive metabolites into CO2 was distinctly different between NPC and SPC except for the 1.7-fold higher utilization of [1-14C]glucose along the oxidative arm of the pentose cycle in the normal cells.     

Metabolic fate of 14C-labelled nicotinamide and adenine in germinating propagules of the mangrove Bruguiera gymnorrhiza

We studied the metabolic fate of [carbonyl-14C]nicotinamide and [8-(14)C]adenine in segments taken from young and developing leaves, stem, hypocotyls, and roots of a shoot-root type emerging propagule of the mangrove plant Bruguiera gymnorrhiza. Thin-layer chromatography was used together with a bioimaging analyser system. During 4 h of incubation, incorporation of radioactivity from [carbonyl-14C]nicotinamide into NAD and trigonelline was found in all parts of the propagules; the highest incorporation rates into NAD and trigonelline were found in newly emerged stem and young leaves, respectively. Radioactivity from [8-(14)C]adenine was distributed mainly in the salvage products (adenine nucleotides and RNA), and incorporation was less in catabolites (allantoin, allantoic acid, and CO2). Adenine salvage activity was higher in young leaves and stem than in hypocotyls and roots. Over a short time, the effect of 500 mM NaCl on nicotinamide and adenine metabolism indicated that NaCl inhibits both salvage and degradation activities in roots.     

The pathways of oxalate formation from phenylalanine,tyrosine, tryptophan and ascorbic acid in the rat

The metabolic pathway by which L-[¹⁴C₁]phenylalanine, L-[¹⁴C₁]tyrosine, L-[¹⁴C₁]tryptophan, and L-[¹⁴C₁]ascorbic acid are converted to [¹⁴C]oxalate have been investigated in the male rate. Only [¹⁴C]oxalate was detected in the urine of rats injected with L-[¹⁴C₁]ascorbic acid, but [¹⁴C]-labeled oxalate, glycolate, glyoxylate, glycolaldehyde, glycine, and serine were recovered from the [¹⁴C₁]-labeled aromatic amino acids. DL-Phenyllactate, an inhibitor of glycolic acid oxidase and glycolic acid dehydrogenase, reduced the amount of [¹⁴C]oxalate recovered in the urine of rats given the [¹⁴C₁]-labeled aromatic amino acids, but increased the amount of [¹⁴C]glycolate formed from L-[¹⁴C₁]-phenylalanine and L-[¹⁴C₁]tyrosine and the amount of [¹⁴C]glycolate produced from [¹⁴C₁]tryptophan. Based on the [¹⁴C]labeled intermediates identified and the relative distribution of the radioactivity, it is postulated that phenylalanine and tyrosine are converted to oxalate via glycolate which is oxidized directly to oxalate by glycolic acid dehydrogenase. Tryptophan is metabolized via glyxylate which is oxidized directly to oxalate by glycolic acid oxidase. Neither glycolate, glyoxylate, glycolic acid oxidase or glycolic acid dehydrogenase are involved in the formation of oxalate from ascorbic acid.     

The effects of alkylthioacetic acids (3-thia fatty acids) on fatty acid metabolism in isolated hepatocytes

Long-chain alkylthioacetic acids (3-thia fatty acids) inhibit fatty acid synthesis from [1-14C]acetate in isolated hepatocytes, while fatty acid oxidation is nearly unaffected or even stimulated. Desaturation of [1-14C]stearate (delta 9-desaturase) is also unaffected. [1-14C]Dodecylthioacetic acid (a 3-thia fatty acid) is incorporated in triacylglycerol and in phospholipids more efficiently than [1-14C]palmitate in isolated hepatocytes. The metabolism of [1-14C]dodecylthioacetic acid to acid-soluble products (by omega-oxidation) is slow compared to the oxidation of [1-14C]palmitate. In hepatocytes from adapted rats (rats fed tetradecylthioacetic acid for 4 days) the rate of [1-14C]palmitate oxidation is increased and its rate of esterification is decreased. Stearate desaturation is also decreased. The rate of cyanide-insensitive peroxisomal fatty acid beta-oxidation is several-fold increased. The metabolic effects of long-chain 3-thia fatty acids are discussed and it is concluded that they behave essentially like normal fatty acids except for their slow breakdown due to the sulfur atom in the 3 position, which blocks normal beta-oxidation.     

Alkylthioacetic acids (3-thia fatty acids) are metabolized and excreted as shortened dicarboxylic acids in vivo

The metabolism of 1-14C-labeled long-chain alkylthioacetic acids (3-thia fatty acids) which are blocked for normal beta-oxidation by a sulfur atom in the beta-position has been investigated in vivo. Most of the injected radioactivity (greater than 50%) was excreted in the urine within the first 48 h. The recovered and identified metabolites were all short sulfoxydicarboxylic acids. The main metabolite from dodecylthioacetic acid was carboxypropylsulfoxy acetic acid. Some bis(carboxymethyl)sulfoxide (dithioglycolic acid sulfoxide) was also found. The main metabolite from nonylthioacetic acid was carboxyethylsulfoxyacetic acid. No sulfones were found. Less than 1% of the 1-14C from the dodecylthioacetic acid was recovered in respiratory CO2 and about 3% of the 1-14C from nonylthioacetic acid. [1-14C]Dodecyl-sulfonylacetic acid was recovered almost quantitatively as carboxypropylsulfonylacetic acid in the urine after 3 h. A significant fraction (10% of the dodecylthioacetic acid was recovered in the phospholipids and triacylglycerols from liver and epidymal fat pad 4 h after injection. These experiments show that the alkylthioacetic acids undergo an initial omega-oxidation followed by beta-oxidation to short dicarboxylic acids.     

The effect of feeding rats with partially hydrogenated marine oil or rapeseed oil on the chain shortening of erucic acid in perfused heart.

1. The metabolism of [14(-14)C]erucic acid and [U-14C]palmitic acid was studied in perfused hearts from rats fed diets containing hydrogenated marine oil, rapeseed oil or peanut oil for three weeks. 2. [14C]Erucic acid was shortened to [14C]eicosenoic acid (20 : 1, n -- 9) and [14C]oleic acid (18 : 1, n -- 9) in perfused rat hearts from all diet groups. The rapeseed oil diet caused a three-fold increase and the marine oil diet a four-fold increase in the amount of chain-shortened products recovered in heart lipids at the end of perfusion, compared to peanut oil diet. 3. The content of C16:1, C18:1 and C20:1 fatty acids was increased in heart lipids of rats fed hydrogenated marine oil or rapseed oil diet, compared to peanut oil diet. 4. Feeding hydrogenated marine oil or rapeseed oil to the rats induced a 85% increase in catalase activity, a 20% increase in the activity of cytochrome oxidase and a 30--40% increase in the content of total CoA in the heart compared to rats fed peanut oil diet. 5. It is suggested that [14(-14)C]erucic acid is shortened by the beta-oxidation system of peroxisomes in the heart. The increased chain shortening in the hearts from animals fed rapeseed oil or partially hydrogenated marine oil for three weeks may be an important part of an adaptation process.     

Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.).

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.     

Factors controlling the selection of tissue-specific metabolic pathways in early embryonic cells

Rana pipiens embryos from the mid-blastula to the early gastrula stage were dissociated into cell cultures, and incubated with ¹⁴C-labeled tryptophan. The uptake of the tryptophan by the cells, its incorporation into protein and its metabolism by enzymes of the serotonin and kynurenine pathways were measured as a function of time, tryptophan concentration, and embryonic stage. It was found that the intracellular concentration of tryptophan was a constant fraction of the extracellular level except for a brief period around stage , during which the cells accumulated the amino acid to a higher concentration than in the external medium. The dominant metabolic pathway of tryptophan was a function of the intracellular concentration; at the lowest levels reported here most of the tryptophan was metabolized via the kynurenine pathway; at the highest levels most was metabolized via the serotonin pathway.     

Galactose and glucose metabolism in galactokinase deficient, galactose-1-P-uridyl transferase deficient and normal human fibroblasts.

Despite the genetic interruption of the Leloir pathway both galactosemic patients and galactosemic fibroblasts can convert galactose to CO2 and TCA precipitable products, although at less than the normal rate. These observations stimulated investigations into the identity of the alternative metabolic routes which allows for galactose metabolism in the absence of in vitro galactose-1-P-uridyl transferase. Four lines of galactosemic cells, each without detectable gal-transferase, produced 14CO2 from [1-14C]-galactose (0.094 mumoles in 20 cc of medium) at approximately 39% +/- 16% the rate of transferase positive cells over a 48-hour period. However, galactokinase deficient fibroblasts produced 14CO2 and TCA precipitable products from [1-14C]-galactose or [U-14C]-galactose at only 3% to 9% the rate of normal fibroblasts. Therefore it seems likely that gal-transferase deficient fibroblasts must first synthesize galactose-1-P for further metabolism of galactose.     

Carbon Balance Studies of Glucose Metabolism in Rat Cerebral Cortical Synaptosomes

Synaptosomes were isolated from rat cerebral cortex and incubated with [U-14C]-, [1-14C]- or [6-14C]glucose. Glucose utilization and the metabolic partitioning of glucose carbon in products were determined by isotopic methods. From the data obtained a carbon balance was constructed, showing lactate to be the main product of glucose metabolism, followed by CO2, amino acids and pyruvate. Measuring the release of 14CO2 from glucose labelled in three different positions allowed the construction of a flow diagram of glucose carbon atoms in synaptosomes, which provides information about the contribution of the various pathways of glucose metabolism. Some 2% of glucose utilized was calculated to be degraded via the pentose phosphate pathway. Addition of chlorpromazine, imipramine or haloperidol at concentrations of 10(-5) M reduced glucose utilisation by 30% without changing the distribution pattern of radioactivity in the various products.