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

doi:10.1016/0003-2697(81)90144-5

Analytical Biochemistry

Volume 110, Issue 1, 1 January 1981, Pages 258-266

https://doi.org/10.1016/0003-2697(81)90144-5 Get rights and content

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.

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NADP+ and NADPH were measured by an enzymatic cycling assay using glucose-6-phosphate dehydrogenase (100). ATP was assayed in neutralized PCA extracts using a luciferase method (101). AMP was assayed using the AMP-glo kit (Promega) following the manufacturer's instructions.
Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP₃Rs) and the mitochondrial Ca²⁺ uniporter (MCU) are key proteins that regulate intracellular Ca²⁺ concentration. Mitochondrial Ca²⁺ accumulation activates Ca²⁺-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP₃R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca²⁺ signaling. Our results show that TKO cells (exhibiting total loss of Ca²⁺ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca²⁺ signaling is dispensable for the bioenergetic needs of both IP₃R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.
Consequences of Neonatal Resuscitation with Supplemental Oxygen
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To determine the degree and duration of hypoxia necessary to demonstrate production of cortical lactate, rat pups were randomized to be ventilated with 21% or 5% O2 for up to 60 minutes. At each time point, beginning after 5 minutes exposure to 21% or 5% O2, a whole-brain freezing method of fixation16 with liquid nitrogen was performed and the frontal cortices of the pups were dissected and kept at −80°C. Lactate concentration in tissue, as a measure of cellular energy metabolism, was determined by standard luciferin–luciferase bioluminescence and biochemical assays, respectively, as previously reported.16
There has been considerable controversy surrounding the optimal inspired oxygen concentration for resuscitation of term and preterm infants. We have developed a rat pup model to quantify both physiologic and biochemical parameters associated with normoxic vs. hyperoxic resuscitation. We have confirmed existing human data that hyperoxic resuscitation of rat pups is associated with a significant delay in onset of spontaneous respiratory efforts. Both 40% and 100% inspired oxygen delayed onset of respiratory activity when compared to 21% oxygen. We have also documented, in the rat pup model, that hyperoxic resuscitation is associated with reduced levels of glutathione at 24 hours post resuscitation. The implications of these and other findings for human infants are that term asphyxiated babies can be safely resuscitated in 21% oxygen and that supplementary oxygen can be reserved for non-responders. In contrast, resuscitation of extremely low gestational age infants does appear to require an initial low inspired oxygen concentration (eg, 30%) with subsequent pulse oximetry titration to optimize oxygenation status.
Adenosine treatment delays postischemic hippocampal CA1 loss after cardiac arrest and resuscitation in rats
2006, Brain Research
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The samples of the cortex, brain stem and hippocampus were dissected from frozen lyophilized sections. Levels of ATP and total adenylates (AXP = ATP + ADP + AMP) were analyzed in tissue samples by microquantitative histochemistry assay methods described previously (Lust et al., 1981). Metabolite levels were reported as nanomoles per milligram dry tissue weight.
Resuscitation from cardiac arrest results in reperfusion injury that leads to increased postresuscitation mortality and delayed neuronal death. One of the many consequences of resuscitation from cardiac arrest is a derangement of energy metabolism and the loss of adenylates, impairing the tissue's ability to regain proper energy balance. In this study, we investigated the effects of adenosine (ADO) on the recovery of the brain from 12 min of ischemia using a rat model of cardiac arrest and resuscitation. Compared to the untreated group, treatment with adenosine (7.2 mg/kg) initiated immediately after resuscitation increased the proportion of rats surviving to 4 days and significantly delayed hippocampal CA1 neuronal loss. Brain blood flow was increased significantly in the adenosine-treated rats 1 h after cardiac arrest and resuscitation. Adenosine-treated rats exhibited less edema in cortex, brainstem and hippocampus during the first 48 h of recovery. Adenosine treatment significantly lowered brain temperature during recovery, and a part of the neuroprotective effects of adenosine treatment could be ascribed to adenosine-induced hypothermia. With this dose, adenosine may have a delayed transient effect on the restoration of the adenylate pool (AXP = ATP + ADP + AMP) 24 h after cardiac arrest and resuscitation. Our findings suggested that improved postischemic brain blood flow and ADO-induced hypothermia, rather than adenylate supplementation, may be the two major contributors to the neuroprotective effects of adenosine following cardiac arrest and resuscitation. Although adenosine did not prevent eventual CA1 neuronal loss in the long term, it did delay neuronal loss and promoted long-term survival. Thus, adenosine or specific agonists of adenosine receptors should be evaluated as adjuncts to broaden the window of opportunity in the treatment of the reperfusion injury following cardiac arrest and resuscitation.
Free radical-mediated neurotoxicity may be caused by inhibition of mitochondrial dehydrogenases in vitro and in vivo
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We previously demonstrated that copper facilitated the formation of reactive oxygen species, and inhibited pyruvate dehydrogenase and α-ketoglutarate dehydrogenase in vitro and in animal models of Wilson’s disease in vivo. However, direct Cu²⁺ toxicity has only been demonstrated for Wilson’s disease. We now hypothesize that inhibition of these mitochondrial dehydrogenases might also contribute to many other injuries and disorders that are reactive oxygen species-mediated. We have modeled reactive oxygen species-mediated injuries using inducers of reactive oxygen species such as hydrogen peroxide, ethacrynic acid or menadione, or another redox active metal (Cd²⁺). Here we demonstrated that these toxic exposures were accompanied by an early marked reduction in both pyruvate dehydrogenase and α-ketoglutarate dehydrogenase activities, followed by a decrease in neuronal mitochondrial transmembrane potential and ATP, prior to murine cortical neuronal death. Thiamine (6 mM), and dihydrolipoic acid (50 μM), required cofactors for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (thiamine as thiamine pyrophosphate), attenuated the reactive oxygen species-induced reductions in these enzyme activities, as well as subsequent loss of mitochondrial transmembrane potential and ATP, and neuronal death. We next tested the effect of thiamine supplementation on an in vivo model of reactive oxygen species-mediated injury, transient middle cerebral artery occlusion, and reperfusion in rats. Oral or i.p. thiamine administration reduced the middle cerebral artery occlusion-induced infarct. These data suggest that reactive oxygen species-induced neuronal death may be caused in part by reactive oxygen species-mediated inhibition of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase in vitro and in vivo, and that thiamine or dihydrolipoic acid may constitute potential therapeutic agents not just against Cu²⁺ neurotoxicity, but may reduce neuronal degeneration in the broader range of diseases mediated by free radical stress.
Compromised metabolic recovery following spontaneous spreading depression in the penumbra
2004, Brain Research
Citation Excerpt :
The tissue at the sites of the various electrodes was not sampled to avoid the potential artifact from the trauma of the craniectomy and/or insertion of the probe. The pieces of tissue were extracted in oil-well racks either in 0.02N HCl or 0.02N NaOH and the levels of adenosine triphosphate (ATP), P-creatine (phosphocreatine), glycogen, glucose, lactate, γ-aminobutyric acid (GABA), citrate and inorganic phosphate (Pi) were determined by enzymatic analysis [33,34]. The results from these experiments are presented graphically to show the differences between the metabolic consequences of SD in normal and ischemic brain.
Spreading depression (SD) has been demonstrated following focal ischemia, and the additional workload imposed by SD on a tissue already compromised by a marked reduction in blood flow may contribute to the evolution of irreversible damage in the ischemic penumbra. SD was elicited in one group of rats by injecting KCl directly into a frontal craniectomy and the wave of depolarization was recorded in two craniectomies 3 and 6 mm posterior to the first one. In a second group, the middle cerebral artery was occluded using the monofilament technique and a recording electrode was placed 5 mm lateral to the midline and 0.2 mm posterior to bregma. To determine the metabolic response in the penumbral region of the cortex ipsilateral to the occlusion, brains from both groups were frozen in situ when the deflection of the SD was maximal. The spatial metabolic response of SD in the ischemic cortex was compared to that in the non-ischemic cortex. Coronal sections of the brains were lyophilized, pieces of the dorsolateral cortex were dissected and weighed, and analyzed for ATP, P-creatine, inorganic phosphate (Pi), glucose, glycogen and lactate at varying distances anterior and posterior to the recording electrode. ATP and P-creatine levels were significantly decreased at the wavefront in both groups and the levels recovered after passage of the wavefront in the normal brain, but not in the ischemic brain. Glucose and glycogen levels were significantly decreased and lactate levels significantly increased in the tissue after the passage of the wavefront. While the changes in the glucose-related metabolites persisted during recovery even in anterior portions of the cortex in both groups in the aftermath of the SD, the magnitude of the changes was greater in the penumbra than in the normal cortex. SD appears to impose an equivalent increase in energy demands in control and ischemic brain, but the ability of the penumbra to recover from the insult is compromised. Thus, increasing the energy imbalance in the penumbra after multiple SDs may hasten the deterioration of the energy status of the tissue and eventually contribute to terminal depolarization and cell death, particularly in the penumbra.
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The aim of the present study was to establish whether aniracetam is capable of protecting cultured rat astrocytes against ischemic injury. Treatment of the cultures with aniracetam (1, 10 and 100 mM) during 24 h ischemia simulated in vitro significantly decreased the number of apoptotic cells. The antiapoptotic effects of the drug were confirmed by the increase of intracellular ATP and phosphocreatine (PCr) levels and the inhibition of the caspase-3 activity. Aniracetam also attenuated cellular oxidative stress by decreased production of reactive oxygen species (ROS). These effects were associated with the decrease in levels of c-fos and c-jun mRNA in primary astrocyte cultures exposed to 24 h ischemia. When cultured astrocytes were incubated during 24 h simulated ischemia with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor or PD98059, a mitogen-activated protein (MAP)/extracellular signal regulated kinase (ERK) (MEK) inhibitor, the cell apoptosis was accelerated. This effect was antagonized by adding 100 mM aniracetam to the culture medium. These findings suggest that the protective effect of aniracetam is mediated by PI 3-kinase and MEK pathways in the downstream mechanisms.

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

Abstract

J. Biol. Chem

Anal. Biochem

Anal. Biochem

J. Biol. Chem

Anal. Biochem

J. Biol. Chem

J. Biol. Chem