THE EFFECT OF HYPERCAPNIC ACIDOSIS UPON SOME GLYCOLYTIC AND KREBS CYCLE-ASSOCIATED INTERMEDIATES IN THE RAT BRAIN

Abstract— In order to study the influence of intracellular pH on the carbohydrate metabolism of brain tissue, the concentrations of glucose, glucose-6-phosphate, pyruvate, lactate, citrate, α-oxoglutarate, malate, glutamate, aspartate and ammonia were measured in rats exposed to 6–40% CO₂, for 45 min. Hypercapnia of increasing severity gave rise to progressive increases in the concentrations of glucose, glucose-6-phosphate and ammonium ion and to progressive decreases in the concentrations of all metabolic acids measured. The results fit with aH⁺ inhibition of a rate-limiting step between glucose-6-phosphate and pyruvate, and by inference from the results published by others it may be assumed that this step is the phosphofructokinase reaction. Since the proportionally largest decrease occurred in a α-oxoglutarate, the results might be compatible either with an inhibition of a second rate-limiting step such as isocitrate dehydrogenase, or with a loss of α-oxoglutarate through carboxylation to citrate.     

THE EFFECTS OF INTOXICATING DOSES OF ETHANOL UPON INTERMEDIARY METABOLISM IN RAT BRAIN

Abstract— The effect of acute (8-min) and prolonged (13-h) exposures to high doses of ethanol upon the intermediary metabolites of rat brain has been studied, with the use of a new freezing technique which minimizes post-mortem changes. Injection of ethanol (80 mmol/kg body wt) produced general anaesthesia within 8 min after administration. At this time there were increases in the brain contents of glucose, glucose-6-phosphate and citrate; there was no change in arterial pCO₂. Rats under ethanol anaesthesia for 13 h showed increases in brain contents of glycogen, glucose and glucose 6-phosphate; and decreases in lactate, pyruvate, α-oxoglutarate and malate. Under similar experimental conditions, arterial pCO₂, increased from 37 to 51 Torr. The changes in levels of metabolites after injection of ethanol were similar to those after administration of many volatile anaesthetic agents or elevation of brain CO₂ by other means. Although brain levels of malate and α-oxoglutarate decreased after prolonged exposure to ethanol, the mitochondrial redox state was maintained. Accordingly, the levels of glutamate and aspartate fell in accordance with the law of mass action. The maintenance of the cytoplasmic and mitochondrial redox states in the brain during ethanol intoxication was in marked contrast to the effects on the liver. We suggest that the different effects observed in brain and liver result from the action of ethanol upon the nerve cell membrane in brain, whereas the primary target in liver is alcohol dehydrogenase.     

INFLUENCE OF HYPOXIA ON CEREBRAL ENERGY STATE IN RATS WITH PORTA-CAVAL ANASTOMOSIS

Abstract— In order to evaluate whether porta-caval anastomosis, and the accompanying hyperammonemia, affect the balance between production and utilization of ATP in the brain, organic phosphates and carbohydrate substrates were measured in control and shunted rats exposed to hypoxia (arterial P_o2 about 30 mm Hg). In the shunted animals the cortical ammonia content was about 2.5 times that measured in the controls, and there was a marked accumulation of glutamine. The intracellular lactate concentration was identical in the control and the shunted groups, and the pattern of change in carbohydrate substrates was similar. There were no significant differences in ATP, ADP or AMP between the groups but the shunted group showed a significantly lower phosphocreatine content. However, the fall in phosphocreatine in the shunted group could be related to a decrease in the sum of phosphocreatine and creatine. It is concluded that the shunting procedure does not disturb the balance between energy production and energy utilization in the brain.     

THE EFFECT OF HYPERTHERMIA UPON OXYGEN CONSUMPTION AND UPON ORGANIC PHOSPHATES, GLYCOLYTIC METABOLITES, CITRIC ACID CYCLE INTERMEDIATES AND ASSOCIATED AMINO ACIDS IN RAT CEREBRAL CORTEX

The influence of hyperthermia on cerebral blood flow, cerebral metabolic rate for oxygen and cerebral metabolite levels was studied by increasing body temperature from 37° to 40°C and 42°C in rats under nitrous oxide anaesthesia maintained at constant arterial CO₂ tension. The metabolic rate for oxygen increased by 5-6% per degree centigrade. At 42°C the increase in cerebral blood Row was comparable to that in the metabolic rate. The increased temperatures were not accompanied by changes in organic phosphates (phosphocreatine, ATP, ADP or AMP) or in lactate/pyruvate ratio. There was an increase in the tissue to blood glucose concentration ratio. At steady state, there was an increase in glucose-6-phosphate but no other changes in glycolytic metabolites or citric acid cycle intermediates, and the only change in amino acids studied (glutamate, glutamine, aspartate, alanine and GABA) was an increase in glutamate concentration.     

砷对兰州鲇组织中代谢酶活性及RNA和蛋白质含量影响

采用毒性试验方法,研究了安全浓度（1.288 mg/L）条件下亚砷酸钠（NaAsO2）对兰州鲇（Silurus lanzhouensis）脑、鳃、肝脏、肌肉4种组织中6-磷酸葡萄糖脱氢酶（G-6-PDH）和乳酸脱氢酶（LDH）活性,以及RNA和蛋白质含量的影响。结果表明,染毒21d时,As（Ⅲ）可显著降低4种组织中G-6-PDH和LDH活性、RNA和蛋白质含量（P0.05）。撤毒后21d,除脑和肝组织中蛋白质含量未恢复到对照组水平（P0.05）,肝脏中G-6-PDH活性超过了对照组水平（P0.05）外,其余各组织中G-6-PDH和LDH活性、RNA和蛋白质含量均可恢复到对照组水平（P0.05）。以上结果表明,As（Ⅲ）对兰州鲇组织中代谢酶活性具有明显的抑制作用,可致组织细胞RNA损伤和可溶性蛋白质减少,但这种影响是可逆的,撤毒后一定时间内可恢复到正常水平。       

Ethanol-induced alterations of the antioxidant defense system in rat kidney

We report here the effects of chronic ethanol consumption on the antioxidant defense system in rat kidney. Thirty-two male Wistar rats were randomly divided in two identical groups and were treated as follows: control group (water for fluid) and the ethanol-fed group (2 g/kg body weight/24 h). The animals were sacrificed after 10 weeks, and respectively 30 weeks of ethanol consumption, and the renal tissue was isolated and analyzed. Results revealed that kidney alcohol dehydrogenase activities increased significantly after ethanol administration, but the electrophoretic pattern of alcohol dehydrogenase isoforms was unmodified. The SDS polyacrylamidegel electrophoretic study of kidney proteins has revealed the appearance of two new protein bands after long-term ethanol consumption. The kidney reduced glutathione/oxidized glutathione ratio decreased, indicating an oxidative stress response due to ethanol ingestion. The malondialdehyde contents and xanthine oxidase activities were unchanged. The antioxidant enzymatic defense system showed a different response during the two periods of ethanol administration. After 10 weeks, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase were activated, while superoxide dismutase, glutathione transferase, and gamma-glutamyltranspeptidase levels were stationary. After 30 weeks, superoxide dismutase and glutathione peroxidase activities were unmodified, but catalase, glutathione transferase, gamma-glutamyltranspeptidase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities were significantly increased. Remarkable changes have been registered after 30 weeks of ethanol administration for glutathione reductase and glucose-6-phosphate dehydrogenase activities, including an increase by 106 and 216' of control values, respectively. These results showed specific changes in rat kidney antioxidant system and glutathione status as a consequence of long-term ethanol administration.     

Potential biomarkers of dichlorvos induced neuronal injury in rats

The present study was designed to develop suitable biochemical markers of chronic dichlorvos exposure using rat as the animal model. Animals were exposed to dichlorvos (6 mg kg-1 (body weight) day-1) for 8 weeks and the activities of five potential markers were assayed. Acetylcholinesterase, assayed as an index of cholinergic function, was found to decrease in both haemolysate and brain tissue. Cytochrome oxidase, used as a marker of impaired energy metabolism, was also seen to decrease in platelets and brains of dichlorvos-treated animals. However, acid phosphatase, a lysosomal marker of tissue injury, was increased in both serum and brains of experimental animals. Chronic dichlorvos exposure also led to a decrease in the activity of glucose-6-phosphate dehydrogenase, which was assayed in brain as an index of oxidative stress. Dichlorvos administration did not affect 2', 3'-cyclic nucleotide phosphohydrolase. The present study therefore, indicates that apart from acetylcholinesterase, which is probably a non-specific marker of dichlorvos neurotoxicity, the levels of cytochrome oxidase, acid phosphatase and glucose-6-phosphate dehydrogenase may serve as useful determinants of dichlorvosinduced neuronal injury.     

Key enzymes of carbohydrate metabolism as targets of the 11.5-kDa Zn(2+)-binding protein (parathymosin)

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.     

A Quantitative Cytochemical Study of Glutamate and Glucose-6-Phosphate Dehydrogenase Activities during Chilling Injury in Tubers of Dioscorea rotundata Poir.

Onyia, G. O. C. and Gahan, P. B. 1985. A quantitative cytochemicalstudy of glutamate and glucose-6-phosphate dehydrogenase activitiesduring chilling injury in tubers of Dioscorea rotundala Poir.—J.exp. Bot. 36: 1249–1256. The response of glucose-6-phosphate dehydrogenase and glutamatedehydrogenase activities in healthy Jamaican Dioscorea rotundalatubers and those chilled at 3 ?C for 1,2,3,4, and 7 d at 70%r.h. were assessed by quantitative cytochemical assays. Bothenzymes in chill-damaged tuber tissues showed a substantiallyhigher activity than did those of the healthy tubers. An early,sharp increase in the response of the NADP-tetrazolium reductasesystem of damaged tuber tissue was significantly higher (P =0.001) than that of healthy tubers or those chilled but ableto recover. This response may be used as an early marker ofchilling injury in the yam tuber. Key words: Dioscorea rotundata Poir, quantitative cytochemistry, yam tuber, glucose-6-phosphate, dehydrogenase, glutamate dehydrogenase, NADPitetrazolium reductase     

THE MITOCHONDRIAL REDOX STATE OF RAT BRAIN

The use of the glutamate dehydrogenase (EC 1.4.1.3) and β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) reactions for the calculation of the mitochondrial redox state of brain has been examined. To prevent post-mortem anoxic metabolism, brains were frozen in less than a second by using a new technique. Levels of ketone bodies in brain were so low relative to the contamination by blood and extracellular fluid that calculation of the mitochondrial redox state using the β-hydroxybutyrate dehydrogenase reaction was not practical. The concentrations of the non-nucleotide substrates of the glutamate dehydrogenase reaction could be accurately measured in brain and themitochondrial [NAD⁺]/[NADH] ratio calculated from the ratio [α-oxoglutarate] [NH₄⁺]/[glutamate]. The calculation is valid if the ratio [α-oxoglutarate] [NH₄⁺]/[glutamate] in mitochondria is the same as that measured in whole tissue. The evidence supporting this conclusion is the near-equilibrium of the aspartate aminotransferase (EC 2.6.1.l) reaction in brain and the observation by others that the distribution of label between α-oxoglutarate and glutamate in brain, after administration of labelled precursors, conforms to expectation. The alanine aminotransferase (EC 2.6.1.2) reaction was not near equilibrium in brain, probably because of the low in vivo activity of the enzyme.     

EFFECT OF HYPOTHERMIA UPON ORGANIC PHOSPHATES, GLYCOLYTIC METABOLITES, CITRIC ACID CYCLE INTERMEDIATES AND ASSOCIATED AMINO ACIDS IN RAT CEREBRAL CORTEX

—The influence of hypothermia upon the metabolism of the brain was studied by reducing body temperature in N₂O-anaesthetized rats to 32, 27 or 22°C, with subsequent measurements of organic phosphates, glycolytic metabolites, citric acid cycle intermediates and associated amino acids. Hypothermia was maintained for either 1 or 2 h and the effect of anaesthesia was evaluated by maintaining unanaesthetized animals at 22°C. Hypothermia had no influence on the cerebral cortical concentrations of ATP, ADP or AMP and there was only a small increase in phosphocreatine. Since the tissue concentrations of glucose and glycogen were reduced, it is concluded that the well known resistance of the hypothermie brain to ischaemia is unrelated to increased energy stores. Hypothermia was accompanied by decreases in the tissue concentrations of fructose-1,6-diphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, pyruvate, lactate, α-ketoglutarate, succinate and malate, but not of glucose-6-phosphate or citrate. These results indicate that metabolic flux is retarded mainly at the phosphofructokinase and isocitrate dehydrogenase steps. The largest relative reduction was seen in α-ketoglutarate, which was possibly secondary to accumulation of ammonia. There was no change in GABA, but a decrease in glutamate and increases in aspartate and alanine. These, changes are compatible with shifts in the aspartate and alanine aminotransferase reactions, possibly induced by the fall in α-ketoglutarate.     

Diversity of Metabolic Patterns in Human Brain Tumors: Enzymes of Energy Metabolism and Related Metabolites and Cofactors

Biopsies from 15 human gliomas, five meningiomas, four Schwannomas, one medulloblastoma, and four normal brain areas were analyzed for 12 enzymes of energy metabolism and 12 related metabolites and cofactors. Samples, 0.01-0.25 microgram dry weight, were dissected from freeze-dried microtome sections to permit all the assays on a given specimen to be made, as far as possible, on nonnecrotic pure tumor tissue from the same region. Great diversity was found with regard to both enzyme activities and metabolite levels among individual tumors, but the following generalities can be made. Activities of hexokinase, phosphorylase, phosphofructokinase, glycerophosphate dehydrogenase, citrate synthase, and malate dehydrogenase levels were usually lower than in brain; glycogen synthase and glucose-6-phosphate dehydrogenase were usually higher; and the averages for pyruvate kinase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and beta-hydroxyacyl coenzyme A dehydrogenase were not greatly different from brain. Levels of eight of the 12 enzymes were distinctly lower among the Schwannomas than in the other two groups. Average levels of glucose-6-phosphate, lactate, pyruvate, and uridine diphosphoglucose were more than twice those of brain; 6-phosphogluconate and citrate were about 70% higher than in brain; glucose, glycogen, glycerol-1-phosphate, and malate averages ranged from 104% to 127% of brain; and fructose-1,6-bisphosphate and glucose-1,6-bisphosphate levels were on the average 50% and 70% those of brain, respectively.     

Evidence for a higher glycolytic than oxidative metabolic activity in white matter of rat brain

Different values exist for glucose metabolism in white matter; it appears higher when measured as accumulation of 2-deoxyglucose than when measured as formation of glutamate from isotopically labeled glucose, possibly because the two methods reflect glycolytic and tricarboxylic acid (TCA) cycle activities, respectively. We compared glycolytic and TCA cycle activity in rat white structures (corpus callosum, fimbria, and optic nerve) to activities in parietal cortex, which has a tight glycolytic-oxidative coupling. White structures had an uptake of [(3)H]2-deoxyglucose in vivo and activities of hexokinase, glucose-6-phosphate isomerase, and lactate dehydrogenase that were 40-50% of values in parietal cortex. In contrast, formation of aspartate from [U-(14)C]glucose in awake rats (which reflects the passage of (14)C through the whole TCA cycle) and activities of pyruvate dehydrogenase, citrate synthase, alpha-ketoglutarate dehydrogenase, and fumarase in white structures were 10-23% of cortical values, optic nerve showing the lowest values. The data suggest a higher glycolytic than oxidative metabolism in white matter, possibly leading to surplus formation of pyruvate or lactate. Phosphoglucomutase activity, which interconverts glucose-6-phosphate and glucose-1-phosphate, was similar in white structures and parietal cortex ( approximately 3 nmol/mg tissue/min), in spite of the lower glucose uptake in the former, suggesting that a larger fraction of glucose is converted into glucose-1-phosphate in white than in gray matter. However, the white matter glycogen synthase level was only 20-40% of that in cortex, suggesting that not all glucose-1-phosphate is destined for glycogen formation.     

甘蓝型油菜子油分的积累与某些生理变化关系的研究

油菜种子发育过程中,其内部的生理代谢过程发生了规律性的变化。伴随着种子的发育进程,６－磷酸葡萄糖脱氢酶、异柠檬酸裂解酶、异柠檬酸脱氢酶和琥珀酸脱氢酶的活性均有不同程度的增强。在油分旺盛合成期,６－磷酸葡萄糖脱氢酶和异柠檬酸裂解酶的活性均达到了最大值,而此时,异柠檬酸脱氢酶和琥珀酸脱氢酶的活属于匀增加较慢;在种子的不同发育时期,高含油量品系的６－磷酸葡萄糖脱氢酶和异柠檬酸裂解酶的活性均高于低含油量的     

Cytoplasmic NADP-linked dehydrogenase activities in avian tissues

Cytoplasmic activities of NADP-linked malic enzyme (E.C. 1.1.1.40), glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) and NADP-linked isocitrate dehydrogenase (E.C. 1.1.1.42) were determined in tissues of selected avian species, and compared with those in mammals. Malic enzyme was generally more active in avian liver and kidney than in the corresponding mammalian tissues. Hepatic activities as high as 200 units/g wet wt and 100 units/g wet wt were recorded in the Nectariniidae and the Ploceidae respectively. Glucose-6-phosphate dehydrogenase was generally less active in avian tissues than malic enzyme. In passerine birds activities were very low indeed, and in most cases spectrophotometrically undetectable. Malic enzyme and glucose-6-phosphate dehydrogenase were highly active in the adipose tissue of mammals but were inactive in the adipose tissue of birds. Marked increases in hepatic malic enzyme and glucose-6-phosphate dehydrogenase activities were associated in birds with premigratory fattening. Activities of isocitrate dehydrogenase were comparable in the corresponding avian and mammalian tissues, including adipose tissue.     

Studies on the function of proplastids in the metabolism of in vitro-cultured tobacco cells III. Source of reducing power for amino acid synthesis from nitrite

Evidence to show the presence of glucose-6-phosphate dehydrogenase,6-phospho-gluconate dehydrogenase, and NADP-dependent malicenzyme in proplastids of in vitro-cultured tobacco cells wasobtained. Amino acid synthesis from nitrite and 2-oxoglutaratein the proplastids was stimulated by addition of 20 mM glucose-6-phosphate.6-Phosphogluconate, malate, and isocitrate did not affect thesynthesis. Nitrite reduction and glutamate synthesis in theproplastids are assumed to be supplied with NADPH₂ as the sourceof reducing power through the reactions catalyzed by glucose-6-phosphatedehyrdogenase and 6-phosphoglyconate dehydrogenase. (Received March 22, 1977; )     

The effects of undernutrition upon the energy reserve of the brain and upon other selected metabolic intermediates in brains and livers of infant rats

—Rats undernourished from the first to the ninth day of life exhibited no decrease in the energy reserve (P-creatine, ATP, glucose and glycogen) of the brain, although they underwent a 41 per cent decrease in body weight. The apparent increase in the cerebral levels of glucose-6-phosphate and the decreases in hepatic glucose and lactate in the starved animals were probably a consequence of the fact that they froze faster than the control animals rather than of any essential differences in vivo. However, decreases in cerebral glutamate (11 per cent) and hepatic glutamate (33 per cent) in the undernourished animals cannot be explained on this basis. Possible explanations for this decrease in cerebral glutamate content are: a decreased supply of glutamate from the liver, a decreased synthesis of glutamate by the brain, or an increased use of glutamate as an energy source. Since levels of glutamate in the brain increase progressively during the first weeks of life, another interesting possibility is that the lower level of cerebral glutamate in undernourished rats represents a biochemical indicator of a delay in the maturation of specific morphological components which are rich in glutamate and are characteristic of the brain.     

Formation of amino acid from urea and ammonia by sequential enzyme reaction using a microencapsulated multi-enzyme system

A microencapsulated multi-enzyme system has been used for the conversion of urea and ammonia into an amino acid, glutamate. The microencapsulated multi-enzyme system contains urease (E.C.3.5.1.5), glutamate dehydrogenase (E.C.1.4.1.3), and glucose-6-phosphate dehydrogenase (E.C.1.1.1.49). The conversion of urea into glutamate is achieved by the sequential reaction of urease and glutamate dehydrogenase; while glutamate dehydrogenase and glucose-6-phosphate dehydrogenase allow for the cyclic regeneration of NADP⁺:NADPH required for the reaction. The rate of production of glutamate is 1.3 μmole per min per ml of microcapsules. The encapsulated multi-enzyme system thus allows for the sequential enzyme reaction for the conversion of urea and ammonia into an amino acid.     

One step purification of glucose-6-phosphate dehydrogenase from brain areas by immunoaffinity chromatography

Glucose-6-phosphate dehydrogenase was purified from rabbit brain cortex using a single immunoaffinity chromatographic step and was contaminated only by a 50 kDa protein. The proteins, separated by SDS-PAGE, were sequenced: the glucose-6-phosphate dehydrogenase was blocked at the N-terminal, the co-eluted protein was similar to -tubulin. Our technique can be applied to purification and sequencing of the enzyme from brain areas or to measure its turnover rate in cultured cells.     

Ammonia release and binding processes and the activity of acid proteinase and glycolysis enzymes in the dynamics of experimental toxic adrenal encephalopathy

The content of ammonium, glutamine, glutamate, aspartate and GABA, glutamine synthetase activity, acid proteinase, hexonase, phosphohexoisomerase and dehydrogenase glucose-6-phosphate were studied in dog brain homogenates after individual injections of Bacillus coli endotoxin (10 micrograms/kg) and adrenaline (75 micrograms/kg) into veins and their combined injections into the carotid artery. Isolated injections of endotoxin and adrenaline were shown to cause transient metabolic compensatory changes. Combined injections caused stable progressing brain metabolic disorders. It is suggested that neurochemical changes influence endogenous development of toxic adrenal encephalopathy.