DECARBOXYLATION STUDIES OF GLUTAMATE, GLUTAMINE, AND ASPARTATE FROM BRAIN LABELLED WITH [1-14C] ACETATE, l-[U-14C]-ASPARTATE, AND l-[U-14C]GLUTAMATE

Studies in vivo and in vitro of the distribution of label in C-1 of glutamate and glutamine and C-4 of aspartate in the free amino acids of brain were carried out. [1-¹⁴C]-Acetate was used both in vivo and in vitro and l -[U-¹⁴C]aspartate and l -[U-¹⁴C]glutamate were used in vitro.

• 1 The results obtained with labelled acetate and aspartate suggest that CO₂ and a 3-carbon acid may exchange at different rates on a CO_a-fixing enzyme.
• 2 The apparent cycling times of both glutamate and glutamine show fast components measured in minutes and slow components measured in hours.
• 3 With [1-¹⁴C]acetate in vitro glutamine is more rapidly labelled in C-1 than is glutamate at early time points; the curves cross over at about 7 min.
• 4 The results support and extend the concept of metabolic compartmentation of amino acid metabolism in brain.

     

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect ¹⁴CO₂ generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced ¹⁴C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [¹⁴C]glutamate and [¹⁴C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-¹⁴C]glutamate and [U-¹⁴C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [¹⁴C]glutamine in the interstitial space when [¹⁴C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

INCORPORATION OF 14C FROM [U-14C]GLUCOSE INTO FREE AMINO ACIDS IN MOUSE BRAIN REGIONS UNDER CYANIDE INTOXICATION

—During anoxia induced by the administration of potassium cyanide, [U-¹⁴C]glucose was injected intraperitoneally into adult mice and they were decapitated at 5, 15 and 30 min after the injection. After freeze-drying in vacuo, differences in the uptake of radioactive carbon from [U-¹⁴C]glucose into free amino acids (glutamate + glutamine, aspartate + asparagine, GABA, alanine and glycine) in mouse cerebral neocortex, cerebellar hemisphere, caudate nucleus, thalamus, hypothalamus and medulla oblongata were investigated (by macroautoradiography and GLC separation) and compared with those obtained under normal conditions. (1) During anoxia, autoradiographical densities in the thalamus and medulla oblongata were higher than that in the cerebral neocortex and caudate nucleus. (2) Among specific radioactivities (d.p.m./μmol) of free amino acids, alanine gave the highest value during anoxia, except in the cerebellar hemisphere and hypothalamus at 5 min and the medulla oblongata at 30 min. (3) During anoxia, the specific radioactivities of alanine and glycine in each brain region did not significantly decrease at 15 and 30 min compared with those under normal conditions. During anoxia, the specific radioactivity of glutamate + glutamine in the cerebellar hemisphere and hypothalamus did not significantly decrease compared with the normal conditions, while that of GABA, aspartate + asparagine and glutamate + glutamine in the cerebral neocortex, caudate nucleus, thalamus and medulla oblongata showed an increase. (4) The percentage decrease of glutamate + glutamine and aspartate + asparagine at 5 and 15 min was highly significant in the cerebral neocortex and caudate nucleus.     

INCORPORATION OF 14C FROM [U-14C]GLUCOSE INTO FREE AMINO ACIDS IN MOUSE BRAIN LOCI IN VIVO UNDER NORMAL CONDITIONS

By macroautoradiography and by GLC separation, differences in the uptake of radioactive carbon from [U-¹⁴C]glucose into free amino acids (glutamate + glutamine, aspartate + asparagine, GABA, alanine and glycine) in mouse cerebral neocortex, hippocampus, thalamus and hypothalamus were investigated. (1) The autoradiographical densities in the thalamus, cerebral neocortex and hippocampus measured with a microdensitometer were higher than that in the hypothalamus at 5 min after subcutaneous injection. At 180 min, densities in the cerebral neocortex, hippocampus and hypothalamus were higher than that in thalamus. (2) The free amino acid levels determined by GLC varied with each brain region. (3) The specific radioactivity (d.p.m./μmol) of alanine in each brain region was higher than that of the other amino acids at 5 min after the injection. The specific radioactivity of GABA in the brain regions was clearly higher than that of (glutamate + glutamine), (aspartate + asparagine) and glycine at 5 and 15 min. (4) The autoradiographical data were in good agreement with the chemical data at 5 min but were different at 180 min. (5) Variations in specific radioactivity of each free amino acid among brain regions at 5 min were influenced greatly by existing free amino acid concentrations in each region.     

THE INFLUENCE OF INTRAVENTRICULARLY INJECTED AMINO ACID EXCITANTS ON THE LABELLING OF ENDOGENOUS BRAIN AMINO ACIDS FROM [U-14C]ACETATE IN NEMBUTALIZED MICE

Mice were anaesthetized with nembutal and the effects of intraventricularly injected excitant amino acids on [U-¹⁴C]acetate metabolism were investigated. The natural excitant amino acids, l -glutamate and l -aspartate, reduced the incorporation of ¹⁴C from [U-¹⁴C]acetate into glutamine, GAB A and possibly alanine. The synthetic excitant amino acid, N-methyl-d -aspartate caused a reduction in the incorporation of ¹⁴C from intraventricularly injected [U-¹⁴C]acetate into all of the brain amino acids labelled by [U-¹⁴C]acetate within 5 min. It is suggested that these effects may be due to changes in pool sizes of tricarboxylic cycle intermediates, to inhibition of acetyl-CoA formation, or both. Differences in the metabolic effects of the synthetic and natural excitants are interpreted in terms of the uptake of the natural amino acids into glutamine-forming pool(s) of glutamate metabolism.     

METABOLISM OF GLUCOSE, AMINO ACIDS, AND SOME RELATED METABOLITES IN THE BRAIN OF TOADS (BUFO BOREAS) ADAPTED TO FRESH WATER OR HYPEROSMOTIC ENVIRONMENTS

The levels and specific radioactivities (SA) of glucose, lactate, pyruvate, α-oxoglutarate and seven amino acids in the brain of toads adapted to fresh water or to an hyperosmotic environment were analysed at various times (5 min–4 h) after an injection of [U-¹⁴C]glucose into the bloodstream. The concentrations and SA of glucose, lactate and five amino acids in blood plasma also were measured. In addition, the SA of glutamine, glutamate, aspartate and GABA in brain were determined 30 min after an injection of [1,5-¹⁴C]citrate into the cisterna magna. The flow of labelled carbon atoms from glucose to amino acids and related metabolites in the toad brain was qualitatively similar to that in the mammalian brain, but quantitatively less than one-tenth of the rate in the brain of rats. Hyperosmotic adaptation induced a large increase in the levels of glucose and amino acids in the brain without affecting the rate of glucose utilization. The SA of several amino acids relative to the SA of glucose were initially lower in hyperosmotically-adapted toads than in toads adapted to fresh water, presumably because of a greater dilution of isotope by the larger amino acid pools in the hyperosmotically-adapted toads. The rates of synthesis of alanine and glutamine from pyruvate and glutamate, respectively, appeared to increase with hyperosmotic adaptation, but the rate of GABA synthesis from glutamate was unaltered. The SA of α-oxoglutarate and glutamate were similar at all time periods in both groups of toads, an indication that these compounds were interconverted much more rapidly than the rate at which α-oxoglutarate was formed from isocitrate. The SA of lactate in comparison to that of glucose varied but was always considerably lower, even at 4 h after the [¹⁴C]glucose injection. After[U-¹⁴C]glucose, glutamine had a SA lower than that of glutamate, whereas after the injection of [1⁴C]citrate, glutamine was formed with a SA much higher than that of glutamate. Hence, glutamate in the toad brain exhibited metabolic compartmentation similar to that in rat brain.     

14C-LABELLED AMINO ACIDS AND GLUCOSE IN RAT BRAIN AND LIVER AFTER INJECTION OF [2-14C]PROPIONATE

Abstract— [2-¹⁴C]Propionate injected into rats was metabolized into [¹⁴C]glucose and ¹⁴C-labelled aspartate, glutamate, glutamine and alanine. The results are consistent with the conversion of propionate into succinate and the oxidation of succinate into oxaloacetate, the precursor of labelled amino acids and the substrate for gluconeogenesis.
The ratio of the specific radioactivity of glutamine to glutamate was greater than 1 during the 30 min period in the brain, indicating that propionate taken up by the brain was metabolized mainly in the 'small glutamate compartment' in the brain. The results, therefore, support the previous conclusion (G aitonde , 1975) that the labelling of amino acids by [¹⁴C]propionate formed from [U-¹⁴C>]-threonine in thiamin-deficient rats was metabolized in the 'large glutamate compartment' of the brain.
The specific radioactivity ratio of glutamine to glutamate in the liver was less than 1 during the 10 min period but greater than 1 at 30min. These findings which gave evidence against metabolic compartments of glutamate in the liver, were interpreted as indicative of the entry of blood-borne [¹⁴C]glutamine synthesized in other tissues, e.g. brain. The labelling of amino acids when compared to that after injection of [U-¹⁴C]glucose showed that [2-¹⁴C]propionate was quantitatively a better source of amino acids in the liver. The concentration of some amino acids in the brain and liver was less in the adult than in the young rats, except for alanine and glutathione, where the liver content was more than double that in the adult.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE: DISTRIBUTION IN SUBCELLULAR FRACTIONS AS A FUNCTION OF TIME AFTER INJECTION OF [2-14C]MEVALONIC ACID, SODIUM [2-14C]ACETATE AND [U-14C]GLUCOSE INTO 15-DAY-OLD RATS

The distribution of [¹⁴C]-labelled material into subcellular fractions of 15-day-old rat brain was studied at 2 and 24 h following intraperitoneal and intracerebral injection of [2-¹⁴C]sodium acetate, [U-¹⁴C]glucose and [2-¹⁴C]mevalonic acid respectively. The total quantity of labelled isoprenoids in the brain was, except for glucose, greater when the precursor was administered intracerebrally. The intraperitoneal route was more advantageous in the case of [U-¹⁴C]glucose. The subcellular distribution of both labelled total isoprenoid material and sterol was distinct for each labelled precursor. Intracerebrally injected [U-¹⁴C]glucose at both time periods studied suggested no dominance of labelling in any fraction. After intraperitoneal injection of [U-¹⁴C]glucose the microsomes were more prominently labelled. Both methods of administration of sodium [2-¹⁴C]acetate resulted in heavy labelling of the myelin fraction after 24 h. The total labelled isoprenoids resided mainly in the microsomes 24 h after injection of [2-¹⁴C]mevalonic acid. Labelled sterol was found to be localized more in the myelin and microsomal fractions for all three precursors than was the labelled total isoprenoids. Depending on the type of experiment to be conducted, each of these precursors can give different results, which must be interpreted accordingly.     

METABOLIC COMPARTMENTATION IN THE BRAIN: METABOLISM OF A TRICARBOXYLIC ACID CYCLE INTERMEDIATE, [1,4-14C] SUCCINATE, AFTER INTRACEREBRAL ADMINISTRATION

Abstract— The metabolism of a tricarboxylic acid cycle (cycle) intermediate, [1.4-'¹⁴C]succinate, was studied in the brain at 2 20 min after intracerebral injection. The oxidation of [¹⁴C]succinate was rapid, as shown by the incorporation of ¹⁴C into cycle amino acids which accounted for about 30 per cent and 70 per cent of the tissue -“Cat 2 and 10 min respectively. During the whole experimental period the specific radioactivity of glutamine was about three times higher than that of glutamate. Thus exogenous [¹⁴C]succinate elicited signs of metabolic compartmentation similar to those seen after the administration of short chain fatty acids or amino acids. A computer programme, based on data obtained previously on the metabolic compartmentation of acetate and of glucose in the brain, was used to simulate the kinetics of labelling of cycle amino acids after an input of [1.4-¹⁴C]succinate. The correspondence of the simulated data with the experimental results was good in the first 10 min after injection, although the deviations were significant at later time points. Incorporation of ¹⁴C into GABA was very low (< 1 per cent of the amino acid -¹⁴C) after the injection of [1.4-¹⁴C]succinate. Further, labelled GABA formation was not detected in the decapitated rat brain labelled in vivo with [1.4-¹⁴C]succinate 2 min beforehand. Since the oxidation of [l,4-¹⁴C]succinate via the cycle yields unlabellcd GABA. whereas the reversal of the reactions in the GABA bypath may introduce ¹⁴C from succinate into the GABA pool, the results indicate that this reversal is negligible even under the most favourable conditions, i.e. post mortem when both the NADH/NAD⁺ ratios and [¹⁴C]succinate concentrations arc high. The observations are therefore consistent with the view that glutamate is the predominant and probably the only source of GABA carbon in the brain both in vivo and post mortem.     

Decreased labeling of amino acids by inhibition of the utilization of [3H,14C]glucose via the hexosemonophosphate shunt in rat brain in vivo

Treatment of rats with 6-aminonicotinamide showed a small but significant decrease in the labeling of amino acids in the brain after injection of [³H]acetate. The results of these experiments also gave evidence of the presence of [³H]glucose and [³H]lactate, and an increase in [³H]glucose content in the brain of 6-aminonicotinamide treated rats. To apportion the contribution of [³H]glucose formed by gluconeogenesis from [³H]acetate to the labeling of amino acids a method was formulated based on the measurement of radioactivity of amino acids, lactate and free sugars in brain after injection of [6-³H]glucose or [1-³H]glucose relative to that after co-injection of [U-¹⁴C]glucose or [2-¹⁴C]glucose. In contrast to the expected formation of [1, 6-³H]glucose by gluconeogenesis from [³H]acetate,³H-labeled glucose isolated from brain, blood and liver showed the presence of [6-³H]glucose only. The values corrected for the presence of [6-³H]glucose showed that treatment with 6-aminonicotinamide had no effect on the labeling of amino acids by oxidation of [³H]acetate. These findings indicated that a significant decrease in the labeling of amino acids from [U-¹⁴C]glucose reported previously and again confirmed using [1-³H], [6-³H], [2-¹⁴C] or [U-¹⁴C]glucose in the present investigation was not due to the inhibition of the activities of enzymes of the citric acid cycle. These results support the postulated role of the hexosemonophosphate shunt for the utilization of glucose in providing neurotransmitter amino acids glutamate and -aminobutyrate.Dedicated to Professor K. A. C. Elliott on his 80th birthday.     

METABOLISM OF d-[U-14C]RIBOSE IN RAT TISSUES

Abstract— d -[U-¹⁴C]Ribose injected subcutaneously into the rat enters the blood, liver and brain. At 30 min after injection 40-70 per cent of the radioactivity in the brain was found in amino acids and only 2-6 per cent in free sugars. In contrast, free sugars (mainly glucose) and carboxylic acids accounted for most of the radioactivity in liver and blood. Evidence for the entry of [U-¹⁴C]ribose into the brain was obtained by intracarotid or intravenous injection of [U-¹⁴C]ribose after interrupting the blood supply to the liver and kidney. Under these conditions the radioactivity in the brain was found in amino acids, carboxylic acids and ribose; no significant amount of [¹⁴C]glucose was detected in brain or heart. It is concluded that ribose is metabolized directly in vivo in the brain. d -[U-¹⁴C]Ribose was metabolized also by brain slices in vitro to form ¹⁴C-labelled amino acids and carboxylic acids; the rate was equivalent to the utilization of 0.65 μ mol of ribose/g/h. The specific radioactivity of glutamine and of γ -aminobutyrate was similar to or higher than that of glutamate in the brain. These results are discussed in the context of metabolic compartments.     

Compartmentation of citric acid cycle metabolism in brain: effect of aminooxyacetic acid, ouabain and Ca2+ on the labelling of glutamate, glutamine, aspartate and gaba by [1-14C]acetate, [U-14C]glutamate and [U-14C]asparate

—(1) The effects of aminooxyacetic acid, ouabain and Ca²⁺ on the compartmentation of amino acid metabolism have been studied in slices of brain incubated with sodium-[1-¹⁴C]acetate, l-[U-¹⁴C]glutamate and l-[U-¹⁴C]aspartate as tracer metabolites. (2) Aminooxyacetic acid (10^-3 m) inhibited the labelling of aspartate from [¹⁴C]acetate and [¹⁴C]glutamate, as well as the incorporation of label from [¹⁴C]aspartate into glutamate and glutamine. It also inhibited the labelling of GABA from all three radioactive precursors, as would be anticipated if there was inhibition of several transaminases as well as glutamate decarboxylase. The RSA of glutamine labelled from [1-¹⁴C]acetate was increased. This finding indicated that the glutamate pool which is utilized for glutamine formation is associated with glutamate dehydrogenase, and this enzyme appears to be related to the ‘synthetic tricarboxylic acid cycle’. AOAA exerted its major inhibitory effects on the citric acid‘energy cycle’with which transaminases are associated. (3) Ouabain (10^-5 m) inhibited the labelling of glutamine to a much greater extent than the labelling of glutamate from [1-¹⁴C]acetate. It also caused leakage of amino acids from the tissue into the medium. Its effect on the glutamate–glutamine system was interpreted to be a selective inhibition of the 'synthetic’citric acid cycle. (4) The omission of Ca²⁺ from the incubation medium was associated with formation of glutamine with RSA less than 1·0 when labelled from [U-¹⁴C]glutamate, [U-¹⁴C]aspartate and lower than normal when labelled from [1-¹⁴C]acetate.     

GLUCOSE AND AMINO ACID METABOLISM IN OCTOPUS OPTIC AND VERTICAL LOBES IN VITRO

(1) The metabolism of glucose and amino acids in vitro was compared in the rat cerebral cortex and the optic and vertical lobes of the octopus brain. (2) Specific activities and pool sizes of the five amino acids, glutamate, aspartate, glutamine, alanine and γ-aminobutyric acid (GABA), were determined in octopus and rat brain slices after 2 hr incubation with 10 mm -[U-¹⁴C]glucose, 10 mm -L-[U-¹⁴C]glutamate, and 10mm -^L-[U-¹⁴C]glutamate with added 10 mM-glucose. Amino acid pool sizes were similar in rat and octopus brain, with the exception of alanine, which was higher in the octopus. Generally specific activities were from four- to 20-fold higher in rat brain. With [U-¹⁴C]glucose as substrate, specific activities of GABA and glutamate were highest in rat; those of alanine and glutamine highest in octopus brain. With ^L-[U-¹⁴C]glutamate the specific activities of GABA and aspartate were highest in rat, that of aspartate highest and GABA lowest in octopus. The addition of glucose to ^L-[U-¹⁴C]glutamate as substrate had little effect on the specific activities of any of the amino acids. (3) The uptake of some amino acids was determined by incubation with [U-¹⁴C]amino acids for 2 hr, and ¹⁴CO₂ formation was also measured. The amount of label taken up by octopus was uniformly 20-25 per cent of that found for rat brain. The amount of ¹⁴CO₂, however, differed according to the amino acid. Four times as much ¹⁴CO₂ was generated from alanine by octopus optic lobe and twice as much by the vertical lobe than rat cortex, but from glutamate, only 24 per cent in the optic and 15 per cent in the vertical lobe. No ¹⁴CO₂ was generated from [U-¹⁴C]GABA in the octopus, by contrast with the rat. (4) Activity of some of the enzymes involved in amino acid metabolism was determined in homogenates of rat cortex and octopus optic and vertical lobes, with and without activation by Triton X-100. Enzymic activities in the octopus, with the exception of alanine aminotransferase, were lower than in the rat, and glutamate decarboxylase could not be detected in octopus brain, in the absence of detergent.     

DECREASED METABOLISM IN VIVO OF GLUCOSE INTO AMINO ACIDS OF THE BRAIN OF THIAMINE-DEFICIENT RATS AFTER TREATMENT WITH PYRITHIAMINE

Abstract— Thiamine deficiency produced by administration of pyrithiamine to rats maintained on a thiamine-deficient diet resulted in a marked disturbance in amino acid and glucose levels of the brain. In the two pyrithiamine-treated groups of rats (Expt. A and Expt. B) there was a significant decrease in the levels of glutamate (23%, 9%) and aspartate (42%, 57%), and an increase in the levels of glycine (26%, 27%) in the brain, irrespective of whether the animals showed signs of paralysis (Expt. A) or not (Expt. B). as a result of thiamine deficiency. A significant decrease in the levels of γ-aminobutyrate (22%) and serine (28%) in the brain was also observed in those pyrithiamine-treated rats which showed signs of paralysis (Expt. A). Threonine content increased by 57% in Expt. A and 40% in Expt. B in the brain of pyrithiamine-treated rats, but these changes were not statistically significant. The utilization of [U-¹⁴C]glucose into amino acids decreased and accumulation of glucose and [U-¹⁴C]glucose increased significantly in the brain after injection of [U-¹⁴C]glucose to pyrithiamine-treated rats which showed abnormal neurological symptoms (Expt. A). The decrease in ¹⁴C-content of amino acids was due to decreased conversion of [U-¹⁴C]glucose into alanine, glutamate, glutamine, aspartate and γ-aminobutyrate. The flux of [¹⁴C]glutamate into glutamine and γ-aminobutyrate also decreased significantly only in the brain of animals paralysed on treatment with pyrithiamine. The decrease in the labelling of, amino acids was attributed to a decrease in the activities of pyruvate dehydrogenase and α-oxoglutarate dehydrogenase in the brain of pyrithiamine-treated rats. The measurement of specific radioactivity of glucose, glucose-6-phosphate and lactate also indicated a decrease in the activities of glycolytic enzymes in the brain of pyrithiamine-treated animals in Expt. A only. It was suggested that an alteration in the rate of oxidation in vivo of pyruvate in the brain of thiamine-deficient rats is controlled by the glycolytic enzymes, probably at the hexokinase level. The lack of neurotoxic effect and absence of significant decrease in the metabolism of [U-¹⁴C]glucose in the brain of pyrithiamine-treated animals in Expt. B were probably due to the fact that animals in Expt. B were older and weighed more than those in Expt. A, both at the start and the termination of the experiments.     

ÈVIDENCE FOR THE COMPARTMENTATION OF GLUTAMATE METABOLISM IN ISOLATED RAT RETINA

—Glucose is a major precursor of glutamate and related amino acids in the retina of adult rats. ¹⁴C from labelled glucose appears to gain access to a large glutamate pool, and the resulting specific activity of glutamate labelled from glucose is always higher than that of glutamine or the other amino acids. Radioactive acetate appeared to label a small glutamate pool. The specific activity of glutamine labelled from acetate relative to that of glutamate was always greater than 1.0. Other precursors of the small glutamate pool were found to include glutamate, aspartate, GABA, serine, leucine and sodium bicarbonate. The level of radioactivity present in retinae incubated with [U-¹⁴C]glucose or [1-¹⁴C]sodium acetate was reduced in the presence of 10^?5m -ouabain. Under these conditions, the relative specific activity of glutamine labelled from [1-¹⁴C]sodium acetate was lowered, but it was raised when [U-¹⁴C]glucose was used as substrate. Ouabain also considerably reduced the synthesis of GABA from [1-¹⁴C]sodium acetate. In all cases ouabain caused a fall in the tissue levels of the amino acids. Aminooxyacetic acid (10^?4m ) almost completely abolished the labelling of GABA from both [U-¹⁴C]glucose and [1-¹⁴C]sodium acetate, while the RSA of glutamine labelled from the latter substrate was significantly increased. Aminooxyacetic acid raised the tissue concentration of glutamate, but caused a fall in the tissue concentrations of glutamine, aspartate and GABA. The results suggest that there are separate compartments for the metabolism of glutamate in retina and that these can be modified in different ways by different drugs.     

THE EFFECT OF DEFICIENCY OF THIAMINE ON THE METABOLISM OF [U-14C]GLUCOSE AND [U-14C]RIBOSE AND THE LEVELS OF AMINO ACIDS IN RAT BRAIN

Abstract— The incorporation of ¹⁴C into amino acids of the brain was determined at different times after injection of [U-¹⁴C]glucose and [U-¹⁴C]ribose to rats maintained on thiamine-supplemented and thiamine-deficient diets for 22 days.
The ¹⁴C-content of amino acids in the brain of thiamine-deficient rats decreased at times 2–10 min after injection of [U-¹⁴C]glucose. but it increased at 2 min and decreased at times 5–10 min after injection of [U-¹⁴C]ribose.
The results of labelling of amino acids indicated that the activities in vivo of the thiamine pyrophosphate requiring enzymes, pyruvate oxidase, a-oxoglutarate dehydrogenase and transketolase were similar in the two groups. It was suggested that the observed decrease in the labelling of amino acids was due to one or more of the following factors: (i) a decrease in the activities of glycolytic enzymes catalysing the conversion of glucose into triose phosphate; (ii) a decrease in the transport of substrate to the active site of the enzymes; or (iii) altered neurohistopathology of the brain.
Thiamine deficiency in rats showed a 5% decrease in glutamate ( P < 0–05), 46% decrease in threonine (P < 0001) and 16% increase in glycine ( P < 0–01) content of the brain.     

EFFECTS OF SODIUM FLUOROACETATE ON THE METABOLISM OF N-ACETYLASPARTATE AND ASPARTATE IN MOUSE BRAIN

A subconvulsant dose of sodium fluoroacetate inhibited the metabolic utilization of intracerebrally-administered N-acetyl-l -[U-¹⁴C]asparticacid and the labelling of glutamine from this precursor in mouse brain, but not the labelling of glutamate or aspartate. A convulsant dose also inhibited the utilization of l -[U-¹⁴C]aspartic acid. When intraperitoneal injection of a convulsant dose of sodium fluoroacetate was followed by intracerebral injection of N-acetyl-l -[U-¹⁴C]asparticacid, the levels of N-acetylaspartate, aspartate and glutamate in brain were lowered, while the glutamine content was increased. The specific radioactivity of glutamine relative to that of glutamate was much lower when these compounds were labelled from l -[U-¹⁴C]aspartic acid than when N-acetyl-l -[U-¹⁴C]aspartic acid was used as the precursor. Intracerebral injection of tracer amounts of l -[U-¹⁴C]aspartic acid reduced the content of N-acetylaspartate in brain and raised the glutamine content. Sodium fluoroacetate had no additional effect on the relative specific radioactivity of glutamine or the content of N-acetylaspartate, aspartate, glutamate or glutamine when l -[U-¹⁴C]aspartic acid was the precursor. We consider the results to be consistent with a selective inhibition both by sodium fluoroacetate and by exogenous aspartic acid of the tricarboxylic acid cycle in brain associated with the biosynthesis of glutamine. We suggest that the activity of this pathway may regulate the metabolism of N-acetylaspartate and aspartate.     

Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa

1. The testis of the ram secretes considerable amounts of amino acids (200μmoles/day) into the fluid collected from the efferent ducts. The principal amino acid in this testicular fluid is glutamate, which is present in concentrations about eight times those in testicular lymph or in blood from the internal spermatic vein. 2. The concentration of glutamate in seminal plasma from the tail of the epididymis is about ten times that in testicular fluid, and, though glutamate is the major amino acid in ejaculated seminal plasma, its concentration is less than in epididymal plasma. 3. After the intravenous infusion of [U-¹⁴C]glucose, labelled glutamate was found in the testicular fluid. Radioactivity was also detected in alanine, glycine, serine plus glutamine and aspartate. Alanine had the highest specific activity, about 50% of the specific activity of blood glucose. 4. When [U-¹⁴C]glutamate was infused, the specific activity of glutamate in testicular fluid was only about 2% that in the blood plasma. 5. Testicular and ejaculated ram spermatozoa oxidized both [U-¹⁴C]glutamate and [U-¹⁴C]leucine to a small extent, but neither substrate altered the respiration from endogenous levels. 6. No radioactivity was detected in testicular spermatozoal protein after incubation with [U-¹⁴C]glutamate or [U-¹⁴C]leucine. Small amounts of radioactivity were detected in protein from ejaculated ram spermatozoa after incubation with [U-¹⁴C]glutamate. 7. The carbon of [U-¹⁴C]glucose was incorporated into amino acids by testicular spermatozoa; most of the radioactivity occurred in glutamate.     

The effect of 6-aminonicotinamide on the levels of brain amino acids and glucose,and their labeling with14C after injection of [U-14C] glucose

The brains of rats paralysed at 4 hr after the administration of 6-aminonicotinamide were found to contain decreased levels of glutamate and -aminobutyrate. The glucose content of the brain of the treated rats was several fold higher than in controls. The incorporation of¹⁴C into brain amino acids at 30 min after the injection of [U-¹⁴C]glucose was decreased by 16%: this was attributed to mainly decreased labeling of glutamate and associated amino acids. The results are discussed in the light of previous findings that the administration of 6-aminonicotinamide resulted in the blockade of the direct oxidation of glucose by the pentose phosphate pathway.     

CHANGES WITHIN METABOLIC COMPARTMENTS IN THE BRAINS OF YOUNG RATS INGESTING LEAD

—[2-¹⁴C]Glucose and [³H]acetate were injected simultaneously into 19-day-old rats suckling from mothers fed either a normal diet or a diet containing 4·5% lead acetate. Changes in the rate of conversion of both precursors into amino acids associated with the tricarboxylic acid cycle were observed. [^I4C]Glucose. In the brain of young rats ingesting lead, the specific radioactivity of glutamate, aspartate, γ-aminobutyrate and glutamine were all significantly lowered relative to that of glucose. Glutamine labelling was the most affected. [³H]Acetate. In comparison with controls, the total amount of ³H in either water or acid-soluble constituents of the brain was the same, but the ³H content of the amino acids was significantly reduced in the lead-treated rats. In both groups, glutamine had the highest specific radioactivity but the time courses of the labelling of glutamine were different. In the control the peak incorporation was reached during the first 5 min, whereas in the experimental animals this occurred at about 10 min after the injection of the precursor, and the specific radioactivity even at that time was less than in controls. When compared with controls, the depression in the labelling of glutamine was accompanied at 5 min by an increase in the specific radioactivity of aspartate. In the lead-treated rats the labelling of GABA was also slowed and the time course seemed to follow that of glutamine rather than glutamate. In spite of the differences in the metabolism of [³H]acetate, metabolic compartmentation of glutamate, assessed by a glutamine : glutamate specific radioactivity ratio higher than 1, was evident even in the brain of the lead-treated animals, although the values of the ratio at 5 and 10 min were less than in controls. There was no evidence of a diminished supply of substrates to the brain in lead intoxication. The overall changes would be consistent with a retardation in the biochemical maturation of the brain in terms of development of glucose metabolism and metabolic compartmentation.