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.     

Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation

Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-¹⁴C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1-¹⁴C]- and [6-¹⁴C]glucose, while with [U-¹⁴C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the “carbon skeletons” for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.     

METABOLISM OF MALONIC ACID IN RAT BRAIN AFTER INTRACEREBRAL INJECTION

Labeled malonic acid ([1-¹⁴C] and [2-¹⁴C]) was injected into the left cerebral hemisphere of anesthetized adult rats in order to determine the metabolic fate of this dicarboxylic acid in central nervous tissue. The animals were allowed to survive for 2, 5, 10. 15 or 30min. Blood was sampled from the torcular during the experimental period and labeled metabolites were extracted from the brain after intracardiac perfusion. There was a very rapid efflux of unreacted malonate in the cerebral venous blood. Labeled CO₂ was recovered from the venous blood and the respired air after the injection of [1-¹⁴C]malonate but not after [2-¹⁴C]malonate. The tissue extracts prepared from the brain showed only minimal labeling of fatty acids and sterols. Much higher radioactivity was present in glutamate, glutamine, aspartate, and GABA. The relative specific activities (RSA) of glutamine never rose above 1.00. Aspartate was labeled very rapidly and revealed evidence of ¹⁴CO₂ fixation in addition to labeling through the Krebs cycle. GABA revealed higher RSA after [1-¹⁴C]malonate than after [2-¹⁴C]malonate. Sequential degradations of glutamate and aspartate proved that labeling of these amino acids occurred from [1-¹⁴C] acetyl-CoA and [2-¹⁴C] acetyl-CoA, respectively, via the Krebs cycle. Malonate activation and malonyl-CoA decarboxylation in vivo were similar to experiments with isolated mitochondria. However, labeled malonate was not incorporated into the amino acids of free mitochondria. The results were compared to data obtained after intracerebral injection of [1-¹⁴C]acetate and [2-¹⁴C]acetate.     

The effect of glucagon on the carbon flux from palmitate into glucose, lactate and ketone bodies, studied with isolated hepatocytes

Glucagon induced a rapid (within 3 min) increase in glucose radioactivity and a decrease in the labeling of ketone bodies when isolated hepatocytes were incubated in the presence of [1-14C]palmitate. Simultaneously, the hormone induced a decrease in the levels of pyruvate and Krebs cycle intermediates and an increase in the level of phosphoenolpyruvate (PEP). The glucagon-induced increase in glucose radioactivity was much larger than the simultaneous decrease in lactate labeling. A comparison of the incorporation of labeled carbon from [1-14C]palmitate and [U-14C]palmitate into glucose and CO2 indicates a selective stimulatory action of glucagon on the flux through the phosphoenolpyruvate carboxykinase (PEPCK) reaction.     

Pentose cycle and reducing equivalents in rat mammary-gland slices

1. Slices of mammary gland of lactating rats were incubated with glucose labelled uniformly with (14)C and in positions 1, 2, 3 and 6, and with (3)H in all six positions. Glucose carbon atoms are incorporated into CO(2), fatty acids, lipid glycerol, the glucose and galactose moieties of lactose, lactate, soluble amino acids and proteins. C-3 of glucose appears in fatty acids. The incorporation of (3)H into fatty acids is greatest from [3-(3)H]glucose. (3)H from [5-(3)H]glucose appears, apart from in lactose, nearly all in water. 2. The specific radioactivity of the galactose moiety of lactose from [1-(14)C]- and [6-(14)C]-glucose was less, and that from [2-(14)C]- and [3-(14)C]-glucose more, than that of the glucose moiety. There was no randomization of carbon atoms in the glucose moiety, but it was extensive in galactose. 3. The pentose cycle was calculated from (14)C yields in CO(2) and fatty acids, and from the degradation of galactose from [2-(14)C]glucose. A method for the quantitative determination of the contribution of the pentose cycle, from incorporation into fatty acids from [3-(14)C]glucose, is derived. The rate of the reaction catalysed by hexose 6-phosphate isomerase was calculated from the randomization pattern in galactose. 4. Of the utilized glucose, 10-20% is converted into lactose, 20-30% is metabolized via the pentose cycle and the rest is metabolized via the Embden-Meyerhof pathway. About 10-15% of the triose phosphates and pyruvate is derived via the pentose cycle. 5. The pentose cycle is sufficient to provide 80-100% of the NADPH requirement for fatty acid synthesis. 6. The formation of reducing equivalents in the cytoplasm exceeds that required for reductive biosynthesis. About half of the cytoplasmic reducing equivalents are probably transferred into mitochondria. 7. In the Appendix a concise derivation of the randomization of C-1, C-2 and C-3 as a function of the pentose cycle is described.     

pH Dependence of Histidine Affinity for Blood-Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids

The effects of pH (3.5-7.5) on the brain uptake of histidine by the blood-brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single-pass carotid injection technique. Cationic amino acid ( [14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ( [14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.     

The Mechanism of Acetate and Pyruvate Oxidation by Trypanosoma cruzi

The epimastigote or culture form of Trypanosoma cruzi oxidizes [3-¹⁴C] pyruvate and [2-¹⁴C] acetate to ¹⁴CO₂ without an apparent increase in overall respiration. This oxidation takes place through the tricarboxylic acid cycle as shown by (a) the incorporation of substrate ¹⁴C into cycle intermediates; (b) the earlier liberation of acetate carboxyl carbon as CO₂; and (c) the characteristic intramolecular distribution of pyruvate and acetate carbon atoms in the skeletal carbon of aspartic and glutamic acids. Upon oxidation of [3-¹⁴C] pyruvate and [2-¹⁴C] acetate, two of the products, alanine and glutamic acid, are found to account for more than 50% of incorporated ¹⁴C; labeling of alanine predominates with [3-¹⁴C] pyruvate while labeling of glutamic acid predominates with [2-¹⁴C] acetate. Using [1- or 6-¹⁴C] glucose as substrate, the pattern of ¹⁴C distribution in soluble metabolites closely resembles that obtained with [3-¹⁴C] pyruvate, in accordance with the joint operation of the Embden-Meyerhof pathway and Krebs cycle. The cycle operation depends on electron transport through the mitochondrial respiratory chain, since antimycin A, at a relatively low concentration, inhibits the oxidation of [2-¹⁴C] acetate to ¹⁴CO₂, to the same extent as the parasite respiration. Though functional in T. cruzi epimastigotes, the oxidative role of the Krebs’ cycle is apparently limited by the absence of an efficient oxidative apparatus. The cycle operation does, however, constitute an important source of skeletal carbon for the biosynthesis of amino acids and can contribute to the process of glycogenesis.     

Metabolic and secretory responses of parotid cells to cationic amino acids. Oxidation of the amino acids and interference with the oxidation of D-glucose or endogenous nutrients.

Cationic amino acids were recently found to stimulate amylase release from rat parotid cells. The possible relevance of their oxidative catabolism to such a secretory stimulation was investigated. D-Glucose, which was efficiently metabolized in parotid cells and which augmented O2 uptake above basal value, failed to affect basal or stimulated amylase release. L-Arginine, L-lysine and L-histidine failed to stimulate the oxidation of either exogenous D-[6-14C]glucose or endogenous nutrients in cells pre-labelled with [U-14C]palmitate or L-[U-14C]glutamine. The oxidation of L-[U-14C]arginine, L-[U-14C]ornithine, L-[U-14C]lysine and L-[U-14C]histidine, all tested at a 10 mM concentration, was much lower than that of D-[U-14C]glucose (5.6 mM). These findings argue against the view that the stimulation of amylase release by cationic amino acids would be related to their role as a source of energy in the parotid cells.     

Lipogenesis and cholesterogenesis in the liver of rats after cholesterol loading

Intensity of fatty acids and separate classes of lipids synthesis was studied in vitro in the liver of white rats at loading by cholesterol in the dose of 300 mg/kg once a day during 30 days by incubation of organ homogenate with [6-(14)C] glucose, [2-(14)C] lysine, [1-(14)C] palmitic acid with following determination of radioactivity of fatty acids, phospholipids, cholesterol, acylglycerols radioactivity was investigated. The inhibition of fatty acids and separate classes of lipids synthesis in vitro in the liver of white rats at loading by cholesterol at the use of [6-(14)C] of glucose and [2-(14)C] lysine, as predecessors of fatty acids and lipids and stimulation of lipids synthesis at the use of [1-(14)C] palmitic acid as the predecessor was established. The loading of white rats by cholesterol results in its synthesis inhibition in the liver during incubation of its homogenates with [6-(14)C] glucose and does not influence the cholesterol synthesis during incubation of homogenates with [2-(14)C] lysine and [1-(14)C] palmitic acid. Thus synthesis of fatty acids and their use in the phospholipids and acylglycerols synthesis in the liver of white rats with hypercholesterolemia sharply decreases during incubation of their homogenates with [6-(14)C] glucose and [2-(14)C] lysine, and the synthesis of cholesterol, phospholipids and acylglycerols - increases during incubation with [1-(14)C] palmitic acid.     

In Vitro Synthesis of Dopamine and Noradrenaline in the Isolated Rat Pineal Gland: Day-Night Variations and Effects of Electrical Stimulation

Isolated rat pineal glands were incubated in vitro in a medium containing [14C]dopamine or [14C]tyrosine, and the tissue contents of 14C-labelled and total dopamine and noradrenaline were determined by HPLC followed by electrochemical detection and scintillation spectrometry. During incubation with [14C]dopamine, the labelled amine accumulated in pineal glands and was partially converted into [14C]noradrenaline. Nomifensine, a neuronal amine uptake blocker, largely inhibited the accumulation of [14C]dopamine and the formation of [14C]noradrenaline. These experiments demonstrated dopamine beta-hydroxylase activity in the sympathetic nerves of the pineal gland. During incubation with [14C]tyrosine, formation of [14C]dopamine and [14C]noradrenaline was observed in the pineal tissue, indicating that noradrenaline can also be synthesized from dopamine, endogenously formed in the gland. Electrical stimulation of the stalk region of the pineal gland during incubation with [14C]dopamine enhanced the accumulation of [14C]dopamine and synthesis of [14C]noradrenaline. Electrical stimulation also enhanced the formation of [14C]dopamine during incubation with [14C]tyrosine. Compared to that at midday, the tissue content of endogenous noradrenaline at midnight was enhanced by 50% and that of dopamine by 450%. The in vitro accumulation of [14C]dopamine, as well as the synthesis of [14C]dopamine and [14C]noradrenaline, was also increased at midnight. In conclusion, sympathetic nerves in the rat pineal gland contain tyrosine hydroxylase and dopamine beta-hydroxylase, the two enzymes required for the synthesis of noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Gluconeogenesis from glycine and serine in fasted normal and diabetic rats.

1. Non-anaesthetized normal and diabetic rats were fasted for 1 day, and [U-14C]glycine, or [U-14C]serine, or [U-14C]- plus [3-3H]-glucose was injected intra-arterially. The rates of synthesis de novo/irreversible disposal for glycine, serine and glucose, as well as the contribution of carbon atoms by the amino acids to plasma glucose, were calculated from the integrals of the specific-radioactivity-versus-time curves in plasma. 2. The concentrations of both glycine and serine in blood plasma were lower in diabetic than in fasted normal animals. 3. The rates of synthesis de novo/irreversible disposal of both amino acids tended to be lower in diabetic animals, but the decrease was statistically significant only for serine (14.3 compared with 10.5 mumol/min per kg). 4. Of the carbon atoms of plasma glucose, 2.9% arose from glycine in both fasted normal and diabetic rats, whereas 4.46% of glucose carbon originated from serine in fasted normal and 6.77% in diabetic rats. 5. As judged by their specific radioactivities, plasma serine and glycine exchange carbon atoms rapidly and extensively. 6. It was concluded that the turnover of glycine remains essentially unchanged, whereas that of serine is decreased in diabetic as compared with fasted normal rats. The plasma concentration of both amino acids was lower in diabetic rats. Both glycine and serine are glucogenic. In diabetic rats the contribution of carbon atoms from glycine to glucose increases in direct proportion to the increased glucose turnover, whereas the contribution by serine becomes also proportionally higher.     

Invalidity of Criticisms of the Deoxyglucose Method Based on Alleged Glucose-6-Phosphatase Activity in Brain

The observations made by Sacks et al. [Neurochem. Res. 8, 661-685 (1983)] on which they based their criticisms of the deoxyglucose method have been examined and found to have no relationship to the conclusions drawn by them. (1) The observations of Sacks et al. (1983) of constant concentrations of [14C]deoxyglucose and [14C]deoxyglucose-6-phosphate, predominantly in the form of product, reflects only the postmortem phosphorylation of the precursor during the dissection of the brain in their experiments. When the brains are removed by freeze-blowing, the time courses of the [14C]deoxyglucose and [14C]deoxyglucose-6-phosphate concentrations in brain during the 45 min after the intravenous pulse are close to those predicted by the model of the deoxyglucose method. (2) Their observation of a reversal of the cerebral arteriovenous difference from positive to negative for [14C]deoxyglucose and not for [14C]glucose after an intravenous infusion of either tracer is, contrary to their conclusions, not a reflection of glucose-6-phosphatase activity in brain but the consequence of the different proportions of the rate constants for efflux and phosphorylation for these two hexoses in brain and is fully predicted by the model of the deoxyglucose method. (3) It is experimentally demonstrated that there is no significant arteriovenous difference for glucose-6-phosphate in brain, that infusion of [32P]glucose-6-phosphate results in no labeling of brain, and that the blood-brain barrier is impermeable to glucose-6-phosphate. Glucose-6-phosphate cannot, therefore, cross the blood-brain barrier, and the observation by Sacks and co-workers [J. Appl. Physiol. 24, 817-827 (1968); Neurochem. Res. 8, 661-685 (1983)] of a positive cerebral arteriovenous difference for [14C]glucose-6-phosphate and a negative arteriovenous difference for [14C]glucose cannot possibly reflect glucose-6-phosphatase activity in brain as concluded by them. Each of the criticisms raised by Sacks et al. has been demonstrated to be devoid of validity.     

Transport of Leucine-Enkephalin Across the Blood-Brain Barrier in the Perfused Guinea Pig Brain

Transport of [tyrosyl-3,5-3H]enkephalin-(5-L-leucine) [( 3H]Leu-enkephalin) across the blood-brain barrier was studied in the adult guinea pig, by means of vascular perfusion of the head in vivo. The unidirectional transfer constant (Kin) estimated from the multiple-time uptake data for [3H]Leu-enkephalin ranged from 3.62 X 10(-3) to 3.63 X 10(-3) ml min-1 g-1 in the parietal cortex, caudate nucleus, and hippocampus. Transport of [3H]Leu-enkephalin was not inhibited by unlabelled L-tyrosine (the N-terminal amino acid) at a concentration as high as 5 mM, or by the inhibitor of aminopeptidase activity bacitracin (2 mM), suggesting that there was no enzymatic degradation of peptide at the blood-brain barrier. By contrast, 2 mM unlabelled Leu-enkephalin strongly inhibited the unidirectional blood-to-brain transport of [3H]Leu-enkephalin by 74-78% in the parietal cortex, caudate nucleus, and hippocampus. The tetrapeptide tyrosyl-glycyl-glycyl-phenylalanine (without the C-terminal leucine of Leu-enkephalin), at a concentration of 5 mM, caused a moderate inhibition ranging from 15 to 29% in the brain regions studied, whereas the tetrapeptide glycyl-glycyl-phenylalanyl-leucine (without the N-terminal tyrosine) at 5 mM was without effect on Leu-enkephalin transport. Unidirectional brain uptake of Leu-enkephalin was not altered in the presence of naloxone at a concentration as high as 3 mM (1 mg/ml), suggesting that there is no binding of Leu-enkephalin to opioid receptors at the blood-brain barrier. It is concluded that there is a specific transport mechanism for Leu-enkephalin at the blood-brain barrier in the guinea pig.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reciprocal changes in amino acid metabolism in mammary gland and liver of the lactating rat on starvation and refeeding as indicated by the tissue accumulation of alpha-amino[1-14C]isobutyrate.

Measurements of the tissue accumulation in vivo and in vitro by hepatocytes and mammary-gland acini of alpha-amino[1-14C]isobutyrate ([1-14C]AIB) were compared in virgin and lactating rats. The results indicate the existence of a reciprocal relationship between mammary gland and liver for AIB accumulation that is dependent on the lactational and the nutritional state of the rat. This suggests that amino acids are preferentially directed to the mammary gland during active lactation.     

Failure of Glucose and Branched-Chain Amino Acids to Normalize Brain Glucose Use in Portacaval Shunted Rats

Several abnormalities in brain and plasma amino acid concentrations caused by portacaval shunting in rats return toward normal after 4 days of intravenous infusion with either glucose or glucose with branched-chain amino acids. To assess the effect of such treatment on brain energy metabolism, regional brain glucose use was measured using [14C]glucose and autoradiography, 5 weeks after portacaval shunting. In one experiment intravenous glucose or glucose with branched-chain amino acids was given for 4 days. In a separate experiment the treatment was given orally for 2 weeks, and in addition to glucose use, brain monoamines and amino acids were measured. No other food was provided; the rats had free access to water. Normally fed shunted rats and sham-operated rats served as controls. Both types of oral treatment lowered the high concentrations of tyrosine, phenylalanine, and glutamine in plasma and brain. Glucose without amino acids normalized brain tryptophan. Levels of brain norepinephrine, 5-hydroxytryptamine (serotonin), and 5-hydroxyindoleacetic acid were significantly raised after shunting. Treatment had no effect on norepinephrine but the glucose diet brought the indoles into the normal range. In contrast, neither intravenous nor oral treatment affected brain glucose use, which remained depressed by 25-30% in all brain areas examined.     

Carbohydrate and amino acid metabolism in Tuber borchii mycelium during glucose utilization: a (13)C NMR study

The metabolism of [1-13C]glucose in the vegetative mycelium of the ectomycorrhizal ascomycete Tuber borchii was studied in order to characterize the biochemical pathways for the assimilation of glucose and amino acid biosynthesis. The pathways were characterized using nuclear magnetic resonance spectroscopy in conjunction with [1-13C]glucose labeling. The enzymes of mannitol cycle and ammonium assimilation were also evaluated. The majority of the 13C label was incorporated into mannitol and this polyol was formed via a direct route from absorbed glucose. Amino acid biosynthesis was also an important sink of assimilated carbon and 13C was mainly incorporated into alanine and glutamate. From this intramolecular 13C enrichment, it is concluded that pyruvate, arising from [1-13C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase and pyruvate carboxylase before entering the Krebs cycle. The transfer of 13C-labeled mycelium on [12C]glucose showed that mannitol, alanine, and glutamate carbon were used to synthesize glutamine and arginine that likely play a storage role.     

Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos

The efficiency with which developing sunflower embryos convert substrates into seed storage reserves was determined by labeling embryos with [U-(14)C6]glucose or [U-(14)C5]glutamine and measuring their conversion to CO2, oil, protein and other biomass compounds. The average carbon conversion efficiency was 50%, which contrasts with a value of over 80% previously observed in Brassica napus embryos (Goffman et al., 2005), in which light and the RuBisCO bypass pathway allow more efficient conversion of hexose to oil. Labeling levels after incubating sunflower embryos with [U-(14)C4]malate indicated that some carbon from malate enters the plastidic compartment and contributes to oil synthesis. To test this and to map the underlying pattern of metabolic fluxes, separate experiments were carried out in which embryos were labeled to isotopic steady state using [1-(13)C1]glucose, [2-(13)C1]glucose, or [U-(13)C5]glutamine. The resultant labeling in sugars, starch, fatty acids and amino acids was analyzed by NMR and GC-MS. The fluxes through intermediary metabolism were then quantified by computer-aided modeling. The resulting flux map accounted well for the labeling data, was in good agreement with the observed carbon efficiency, and was further validated by testing for agreement with gas exchange measurements. The map shows that the influx of malate into oil is low and that flux through futile cycles (wasting ATP) is low, which contrasts with the high rates previously determined for growing root tips and heterotrophic cell cultures.     

Metabolism of [14C]citrulline in the perfused sheep and goat udder

1. A lactating-sheep mammary gland was perfused for 12h in the presence of l-[2-(14)C]-citrulline and received adequate quantities of glucose, acetate and amino acids. Two lactating-goat udders were similarly perfused in the presence of either l-[carbamoyl-(14)C,-2-(14)C]citrulline or l-[carbamoyl-(14)C,1-(14)C]citrulline and l-[4-(3)H]arginine. 2. In these experiments, [(14)C]citrulline was substantially oxidized to CO(2) and converted into arginine and proline of casein. 3. The specific radioactivities of arginine, ornithine and proline of the plasma increased after passage through the udders, demonstrating that [(14)C]citrulline is metabolized by the mammary gland. 4. The presence of two unknown radioactive metabolites of [(14)C]citrulline was detected in the perfusate. These substances were not found after incubation in vitro of oxygenated blood in the presence of the radioactive precursor. 5. From these experiments, it is concluded that citrulline is metabolized in mammary tissue by way of arginine to urea, ornithine and proline.     

d-β-HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D-β-hydroxybutyrate (β-OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3-¹⁴C]β-OHB or [2-¹⁴C]glucose was injected subcu-taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3-¹⁴C]β-OHB rapidly labelled brain amino acids at all ages whereas [2-¹⁴C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3-¹⁴C]β-OHB from birth-15 days of age whereas brain alanine was more heavily labelled by [2-¹⁴C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β-OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [¹⁴C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2-^,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3-¹⁴C]β-OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [¹⁴C]label from [2-¹⁴C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [¹⁴C]label into the brain amino acids of the 15-day-old [3-¹⁴C]β-OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?-OHB at 6 days was 19 8 min and at 15 days was 12-2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3-¹⁴C]β-OHB injected rats compared to the [2-¹⁴C]glucose injected rats despite a heavier labelling of brain glutamate in the [3-¹⁴C]β-OHB injected group. We interpreted these data to mean that β-OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.