Janet V. Passonneau

A comparison of three methods of glycogen measurement in tissues

Abstract Three methods have been used for analysis of glycogen in tissue homogenates: hydrolysis of the tissue in acid and followed by enzymic analysis of the resulting glucose; enzymic hydrolysis with amylo-α-1,4-α-1,6-glucosidase, again followed by enzymic measurement of glucose; and degradation of the glycogen with phosphorylase and debrancher complex coupled to measurement of the resulting glucose-1- P . The two enzymic procedures yielded equivalent results with all tissues examined (brain, liver, muscle and polymorphonuclear leucocytes). Acid hydrolysis of the tissues resulted in higher values for brain tissue only, presumably due to the hydrolysis of the gangliosides and cerebrosides present in brain.

CONCENTRATIONS OF ENERGY METABOLITES AND CYCLIC NUCLEOTIDES DURING AND AFTER BILATERAL ISCHEMIA IN THE GERBIL CEREBRAL CORTEX

Abstract— The concentrations of metabolites which reflect energy production or use (P‐creatine, ATP. ADP. 5′AMP, glucose, glycogen and lactate) and cyclic nucleotides (cyclic AMP and cyclic GMP) were measured in gerbil cortex during ischemia and recirculation. Bilateral ischemia of the gerbil brain was chosen as a model to ensure the assessment of short periods of ischemia without ambiguity. The metabolites and cyclic nucleotides were measured after, 1, 5. 20. 30 and 60 min of ischemia; and 1, 5, 30, 60 and 360 min after circulation was reestablished. The greatest changes in metabolites and cyclic nucleotides due to ischemia occurred during the 1st min; ischemia of longer duration had little further effect. However, the restoration of the metabolic profile was altered by the duration of the ischemic period. In general, the longer the period of ischemia, the slower the replenishment of high‐energy phosphate compounds and energy sources. Cyclic AMP increased 5‐ to 13‐fold during ischemia; cyclic GMP decreased to as little as one‐fifth control values 60min after occlusion. During recirculation, cyclic AMP increased as much as 100‐fold, while cyclic GMP increased up to 6‐fold. The temporal derangements in cyclic nucleotide concentrations coincide with the loss and restoration of cortical activity; a possible mechanism has been suggested.

The enzymatic measurement of adenine nucleotides and P-creatine in picomole amounts

Abstract Enzymatic methods are described for the analysis of ATP, ATP + ADP, total adenylates, or P-creatine in biological samples. The methods include (i) direct fluorometric procedures for the measurement of 0.1–10 nmol using hexokinase and glucose-6-P-dehydrogenase as the indicator step; (ii) an enzymatic cycling procedure with a sensitivity of 1–50 pmol; and (iii) the measurement of light emission in the luciferin-luciferase system with a sensitivity of 0.1–80 pmol.

Factors affecting the turnover of cerebral glycogen and limit dextrin in vivo.

The turnover of cerebral glycogen in mice has been investigated by using [U‐14C]glucose as a precursor. The time required for turnover of total glycogen and limit dextrin has been determined in normal animals and animals given phenobarbital or hydrocortisone. In all 3 groups, the turnover time for limit dextrin was twice that of total glycogen. Phenobarbital increased the time for turnover of total glycogen and limit dextrin approximately 2‐fold, whereas hydrocortisone diminished the turnover time of both fractions to one‐half. The accumulation of glycogen during phenobarbital anesthesia (2·5‐fold) is attributed to the decrease in rate of phosphorolysis rather than elevated glycogenesis. The ratio of phosphorylase a to total phosphorylase was significantly decreased in the brains of phenobarbital‐treated mice, while the ratio of glycogen synthetase I to total synthetase activity was not affected. The administration of hydrocortisone had no effect on either the phosphorylase or synthetase of mouse brain. A mathematical model was devised to determine the rate constants for incorporation of labelled glucose into brain glycogen and the subsequent loss of radioactivity. Metabolite levels and enzyme activities have been correlated with the observed changes in glycogen turnover.

Energy metabolism in delayed neuronal death of CA1 neurons of the hippocampus following transient ischemia in the gerbil.

The delayed death of CA1 neurons in the gerbil has been reported to occur at 4 days of reflow following 5 min of bilateral ischemia. Samples of the CA1 and CA3 somal region of the hippocampus, as well as of the parietal cortex, were dissected from frozen dried sections of gerbil brains frozenin situ between 1.5 and 96hr of reflow following 5 min of bilateral ischemia and the concentrations of the adenylates, P-creatine, glucose, glycogen, and lactate were determined. The values for high-energy phosphates were restored by 1.5hr of recirculation in all three regions and remained at or above control in the CA3 region and cortex for up to 96hr. In contrast, the P-creatine and ATP decreased in the CA1 region at 48 and 96hr of reflow, respectively. The total adenylates also decreased in the CA1 region at 96h, but the normal energy charge in this area indicated that the surviving tissue was metabolically viable. A glucose overshoot was exhibited in the three regions at all time periods except 6 and 96 hr. At 6 hr of reflow, there was a transient return of glucose levels toward those of control. By 96 hr, the glucose in the CA3 region and cortex was not significantly different from control but was elevated in the CA1 region. The lactate levels were depressed from 1.5 to 12 hr of recirculation in all areas, but the decrease was significant only in the cerebral cortex. The concentration of glycogen was significantly elevated at 6 hr in all regions, then was restored by 24 to 48 hr, only to increase once again in the affected CA1 region. The results clearly indicate that metabolic perturbations persist for long periods of time after ischemic durations that are compatible with the survival of the animal but that the loss of the CA1 neurons cannot be attributed to a failure in energy metabolism.

ALTERATIONS OF CYCLIC NUCLEOTIDE-RELATED ENZYMES AND ATPase DURING UNILATERAL ISCHEMIA AND RECIRCULATION IN GERBIL CEREBRAL CORTEX

Abstract— Several enzyme activities were determined in gerbil cerebral cortex during unilateral ischemia or in the post‐ischemic period following 1 h of ischemia. Adenylate cyclase and Na + ‐K + ‐activated ATPase showed essentially the same pattern. Neither enzyme changed during ischemia but the activities decreased on recirculation to 40–60% of right side control by 5 h. The ATPase had returned to control level by 20h; the adenylate cyclase by 7 days of recirculation. Particulate cyclic AMP‐dependent protein kinase in the ischemic left hemisphere decreased throughout the 6h of ischemia. It remained depressed in the first 5 h of the post‐ischemic period but returned to control by 20 h. The soluble protein kinase activity, the soluble cyclic AMP and cyclic GMP phosphodiesterase and the Mg2+ dependent ATPase did not change significantly during the ischemic or post‐ischemic periods. The results suggest that ischemia and/or recirculation may affect cellular membranes and membrane‐bound enzymes, in particular. Furthermore, the results imply that despite apparent metabolite recovery during the post‐ischemic period, enzymatic changes are occurring that may be important for both the quality of recovery and the response to further ischemic insult.

Cyclic Adenosine Monophosphate, Metabolites, and Phosphorylase in Neural Tissue: A Comparison of Methods of Fixation

Fixation of rat brain tissue by freeze-blowing, microwave irradiation, immersion of whole rats in liquid nitrogen, and decapitation into liquid nitrogen indicates that postmortem changes in metabolites and enzyme forms are minimal in freeze-blown brains. Cyclic adenosine monophosphate levels are lowest in microwave-irradiated brains, which has been interpreted by some investigators to indicate rapid fixation and minimal anoxia. However, the changes in phosphocreatine, adenosine triphosphate, lactate, and phosphorylase clearly demonstrate that fixation by freeze-blowing or immersion in liquid nitrogen more closely approximate the state in vivo.

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.

POST-ISCHEMIC CHANGES IN CERTAIN METABOLITES FOLLOWING PROLONGED ISCHEMIA IN THE GERBIL CEREBRAL CORTEX

‐Eight metabolites were measured in the post‐ischemic period following either 1 or 3 h of unilateral ischemia in the gerbil cerebral cortex. The levels of ATP, P‐creatine, glucose, glycogen and GABA were essentially restored by 1 h after ischemia. In the 3 h ischemic animals. glycogen continued to increase to greater than control values aftcr 5 and 20 h of recirculation. The Icvels of glutamate were unchanged during the ischemic episode, but decreased to 60% of control at Smin and 1 h after either period of ischemia. The concentrations of cyclic AMP, which were 4‐to 5‐fold elevated during ischemia. increased an additional 6‐fold 5 min after recirculation in both groups. Arter 1 h of recovery. the levels were not different from control values. After the 1 h ischemic period, lactate levels recovered between 5 and 20 h of recirculation. In the 3 h ischemic animals. lactate concentrations were still elevated even after 20 h of recirculation. These data suggest that with the exception of lactate. recovery of metabolites is not sevcrely compromiscd by either 1 or 3 h of ischemia. Furthermore, the changes in glycogen. glutamate and cyclic AMP after recirculation suggest that the recovery process is not just a rcversal of the changes observed during ischemia.

A modified cyclic AMP binding assay.

Abstract A protein binding method based on Gilmans procedure for the measurement of cyclic AMP is described with two particular modifications. First, the assay utilizes a commercial phosphodiesterase preparation which contains a cyclic AMP binding protein as a contaminant; and second, a cation-exchange resin is used for the separation of free from bound cyclic AMP. These modifications serve to reduce both the time and expense of the measurement of cyclic AMP.