Demoy W. Schulz

GLYCOGEN, AMMONIA AND RELATED METABOLITES IN THE BRAIN DURING SEIZURES EVOKED BY METHIONINE SULPHOXIMINE1

Abstract— The levels of ATP, P‐creatine, glucose, glycogen, lactate, glutamate and ammonia were measured in mouse brain after administration of the convulsive agent methionine sulphoximine (MSO). No changes were observed in ATP and P‐creatine levels either before or during the seizures. Lactate levels were unchanged until the onset of seizures (4–5 hr) at which time the levels increased an average of 65 per cent. Glucose and glycogen levels increased progressively. Just before the onset of seizures the levels had increased 95 and 62 per cent, respectively. During the seizures both substances had increased a total of 130 per cent. Comparable changes were found in cerebral cortex, cerebellum and subcortical forebrain. Through the use of quantitative histochemical methods it was found that the greatest increases in glycogen occurred in layers I and III (layers II and IV were not analysed). Progressively smaller changes were found in layers V and VI and no increase at all was found in the subjacent white matter. Glucose, in contrast to glycogen, increased to about the same degree in all cerebral layers and in subjacent white matter.

Control of glycogen levels in brain.

Abstract— Prolonged (6 hr) anaesthesia with phenobarbital in mice or rats results in a doubling or tripling of brain glycogen. Increases were also observed if high levels of plasma glucose were maintained for 6 hr. In alloxan diabetes brain glycogen was not elevated in spite of the high plasma glucose concentrations. However, administration of insulin to such diabetic animals, together with enough glucose to maintain high plasma levels, resulted in at least a doubling of brain glycogen in 6 hr. Phenobarbital can still increase brain glycogen in diabetic animals.

An enzymic method for measurement of glycogen

An enzymic method for measuring glycogen has been described in detail. Glycogen plus inorganic phosphate and TPN+ are converted in one analytical step to 6-P-gluconolactone and TPNH. The TPNH is measured by its fluorescence or ultraviolet absorption. The method uses commercially available enzymes: phosphorylase a, P-glucomutase, and glucose-6-P dehydrogenase. It takes advantage of the fact that most phosphorylase preparations contain sufficient transglucosylase and glucosidase to permit complete degradation of glycogen. The specificity is such that whole tissue can be analyzed directly. The sensitivity is sufficient to measure 0.05 μg of glycogen with precision.

An enzymic method for the measurement of inorganic phosphate

Abstract An enzymic method is described for measuring inorganic phosphate. The successive enzyme steps are those catalyzed by glycogen phosphorylase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase. TPNH is finally produced from TPN + in proportion to the inorganic phosphate present, and is measured by its fluorescence or light absorption. The method has two advantages: ( 1 ) The analytical reaction takes place at neutrality thereby permitting measurement of inorganic phosphate in the presence of very unstable organic phosphates. ( 2 ) The method is very sensitive. In terms of concentration the sensitivity is limited mainly by the degree to which phosphate can be removed from the reagents. In terms of amount, there is no real sensitivity limit since the TPNH formed can be measured by enzymic cycling.

Effect of Chronic Hypernatremic Dehydration and Rapid Rehydration on Brain Carbohydrate, Energy, and Amino Acid Metabolism in Weanling Mice

Abstract: This is a study of the effects of chronic hypernatremic dehydration and rehydration on carbohydrate, energy, and amino acid metabolism in the brains of weanling mice. Chronic hypernatremic dehydration induced by 4 days of water deprivation and salt loading was associated with severe weight loss (no other observed clinical effects), increased brain Na+ levels, and a decreased brain water content. Changes in the concentrations of brain glucose, glycolytic and citric acid cycle metabolic intermediates, and phosphocreatine were compatible with reduced cerebral metabolic rate. In adaptation to chronic hypernatremia, there was a significant increase in the content of the measured brain amino acids. Rapid rehydration over a 4‐h period with 2.5% dextrose in water returned plasma Na+ levels and brain Na+ and water contents to normal. After rehydration, metabolites were altered in a manner consistent with increased fluxes through the glycolytic pathway and citric acid cycle; the brain glycogen content almost tripled. Brain taurine and glutamine levels were not lowered by rehydration, and the total content of the measured amino acids in brain was still significantly higher than in controls. We speculate that these metabolic perturbations may relate to the development of cerebral edema and seizures or coma following rapid rehydration of humans with chronic hypernatremic dehydration.

Effects of Acute, Subacute, and Chronic Diabetes on Carbohydrate and Energy Metabolism in Rat Sciatic Nerve: Relation to Mechanisms of Peripheral Neuropathy

To address the problem of the pathogenesis of diabetic neuropathy, rats were made diabetic by alloxan administration, and sciatic nerves were sampled for electrolyte and water content and levels of selected carbohydrates and intermediates in energy metabolism at 3, 6, and 26 weeks. Significant increases were seen in the nerve content of glucose, sorbitol, and fructose. Decreases of myo-inositol were not statistically significant. Glucose-6-phosphate was increased at all times; fructose-1,6-bisphosphate was elevated at 6 and 26 weeks. Nerve ATP and phosphocreatine levels were both increased concomitantly, as was the energy charge. Nerve lactate levels increased only at 26 weeks when plasma lactate levels were also high. Plasma ketone bodies were elevated throughout the 26-week experimental interval. It is postulated that ketone bodies were being used as alternative metabolic fuels in diabetic nerve, thereby causing inhibition of pyruvate oxidation and increased aerobic production of lactate. Increased plasma ketone body levels could also inhibit hepatic lactate uptake. There was no other evidence for hypoxia/ischemia. Lactate:pyruvate ratios did not differ from control values at any time in these ketotic hypoinsulinemic animals. Five major hypotheses have been proposed to explain the pathogenesis of diabetic neuropathy: 1) hypoxia/ischemia, 2) hyperglycemie pseudohypoxia, 3) myo-inositol deficiency, 4) fructose and polyol accumulation and osmotic disequilibrium, and 5) nonenzymatic glycation of macromolecules by fructose and glucose. The data obtained in this study seem to fit best with hypotheses 4 and perhaps 5.

Effects of acute hyperosmolar NaCl or urea on brain H2O, Na+, K+, carbohydrate, and amino acid metabolism in weanling mice: NaCl induces insulin secretion and hypoglycemia

This study compares early and late effects of the injection of hyperosmolar NaCl and urea of equal osmolarity on selected aspects of brain water, electrolyte, carbohydrate, amino acid, urea, and energy metabolism in normal suckling-weanling mice. One hour after treatment, salt-treated mice were critically ill, while the behavior of urea-treated animals could not be distinguished from that of controls. This clinical difference could not be explained on the basis of differences in plasma osmolality, the brain water content, or the degree of hemorrhagic encephalopathy. The injection of NaCl induced a 14-fold increase in plasma insulin and a progressive fall in the plasma glucose concentration (a reduction of 66% at 1 hr). In contrast, plasma glucose levels in urea-injected mice were unchanged. Prior to the fall in plasma glucose levels, metabolite changes in the brains of NaCl-injected mice were compatible with facilitation of transfer of glucose from the blood to the brain, increased metabolic flux in the Embden-Meyerhof and Krebs citric acid cycle pathways, and increased energy production. With the exception of the glucose content (unchanged), similar metabolite changes were seen in brain soon after urea injection. In the brains of the hypoglycemic NaCl-treated mice, glucose levels were reduced 80%, and glycogen 41%. Other metabolite changes were compatible with decreased glycolysis and metabolic flux through the Krebs citric acid cycle. In contrast, with few exceptions, at a similar time after injection, metabolite levels had returned to normal in the urea-treated mice. Permeability of the brain to urea was also examined. Brain urea reached high levels at 2hr but returned to near baseline at 6hr. Both hyperosmolar solutions increased the brain content of aspartic and glutamic acids 1 hr after injection. The failure of hypoglycemic mice with hypernatremia and elevated plasma osmolality (range, 416–434 mOsm/kg H2O) to respond to 1M glucose (30ml/kg) may have been due to the ill effects of the additional hyperosmolar load. The possibility remains that the encephalopathy induced by hyperosmolar NaCl, but not by hyperosmolar urea, is in some way related to the sudden elevation of brain Na+ and/or Cl− ions.

Inorganic Phosphate Fluorimetric Method

Publisher Summary Inorganic phosphate is a substrate or product of many enzyme reactions and affects the activity of enzymes such as hexokinase, phosphofructokinase, and glucose-6-phosphate dehydrogenase. There are various methods for the determination of phosphate; however, each method has its disadvantages. In the method of Fiske and Subbarow, acid-labile phosphate is hydrolyzed and can be modified, but the analysis is time-consuming and less sensitive. This chapter describes an enzymatic method for the determination of phosphate. The enzymatic method is carried at neutral pH. The sensitivity of the method is limited only by the phosphate contamination of the reagents. The method depends on the phosphorolytic cleavage of glycogen by phosphorylase a. The method finds application in biochemistry and clinical biochemistry. The principle behind the method is that the increase of NADPH, as measured by the fluorescence, is proportional to the amount of Pi. Filter fluorimeter with primary filter for excitation at 360 nm and secondary filter for emitted light of 460 nm is used in the method.

670E VALPROATE MAY DELAY BRAIN MATURATION AND PRODUCES HEPATIC DYSFUNCTION IN INFANT MICE

Normal nursing 2 to 4 d-old mice received 100 or 200 mg/kg of valproate (VAL) s.c. once daily for 5 d. At 100 mg/kg there was no effect on body weight (wt). At 200 mg/kg/, body wt was reduced 20% (P=0.001), brain wt, 20% (P<0.001) and brain [K+], 3% (P=0.009); brain [Na+] tended to rise. Both doses increased brain glycogen (31%), glucose (27%), glucose-6-P (15%), fructose-6-P (21%) and decreased fructose-di-P (35%), pyruvate (14%), citrate (15%), α-ketoglutarate (14%) and malate (18%), suggesting a decreased metabolic flux through the glycolytic pathway and Krebs citric acid cycle. Together with the decrease in P-creatine (7%) the findings suggest reduced cerebral metabolic rate (CMR). Brain aspartate was reduced 12% at 100 mg/kg VAL (P<0.001) and 25% at 200 mg/kg (P=0.002). At 200 mg/kg VAL, brain glutamate fell 14% (P=0.006) while taurine increased 13% (P=0.039). Both doses tended to decrease brain GABA and increase brain glycine. Both VAL doses decreased plasma β-hydroxybutyrate (β-OHB) concentration 60% (P<0.001); plasma fatty acid levels were normal. At 200 mg/kg VAL the liver glycogen content was reduced 73% (P<0.001).VAL-induced decreased brain wt and [K+], the apparent reduction in CMR, and the direction of the amino acid changes are most compatible with delayed brain maturation. Critical reductions in plasma β-OHB and liver glycogen levels may relate to VAL-associated hepatic-deaths in infants and children.