Phosphorus-31 nuclear magnetic resonance spectroscopy of human retinoblastoma cells: correlations with metabolic indices

The ³¹P nuclear magnetic resonance spectrum of cultured human Y-79 retinoblastoma cells was obtained at 121 MHz on intact cells trapped in agarose threads. The spectrum was dominated by monoester peaks, which varied in relative concentration from preparation to preparation. Resonances from phosphocreatine, phosphodiesters and diphosphodiesters also exhibited variability relative to ATP. The main monoester was identified as phosphorylcholine by ³¹P-NMR of perchloric acid extracts. It was determined that the changes in monoester concentration correlated with feeding pattern. Phosphorus spectra of cells 1,2 and 3 days post feeding showed a 40% decrese in the relative concentration of phosphorylcholine concentration over the 3 day period. Phosphocreatine, phosphodiesters and diphosphodiesters increased relative to ATP during the same period. Growth curve experiments and oxygen consumption measurements indicated that the decrease in phosphorylcholine correlated with a decrease in cellular growth and oxygen consumption. We conclude that monoester concentration may be a useful indicator of nutritional status in these cells and possibly in intact tumors.     

Stabilization of a proteolytically sensitive cytoplasmic recombinant protein during transition to downstream processing

The influence of aeration and glucose feeding on the stability of recombinant protein A in Escherichia coli during the transition period from a fed‐batch cultivation to downstream processing was studied. Neither interruption of the feeding under aerobic conditions nor anaerobic conditions in presence of glucose could stabilize protein A completely and the intracellular ATP pool did not decrease to less than 0.75–1 mM by this treatment. On the other hand, the absence of both oxygen and glucose resulted in a decrease of the ATP pool to less than 0.5 mM and almost complete stabilization of protein A. The decrease of ATP was more severe when sulfite was used instead of nitrogen gas to create anaerobic conditions in presence of glucose. This also resulted in nearly complete stabilization of protein A, which might be explained by an inhibiting effect of sodium sulfite on fermentation. Therefore, protein stabilization and decrease of the ATP pool were correlated in experiments in vivo. The concentrations of ADP and AMP increased during starvation and may also play a role in stabilization of the protein in vivo. ATP may be a limiting factor of proteolysis also during further steps of downstream processing. Its concentration decreases by 80–90% during harvesting and centrifugation of biomass and even further during disruption of cells. However, neither addition nor regeneration of ATP in cell disintegrate was enough to restore degradation of protein A, indicating that an additional factor limits proteolysis in vitro. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 730–738, 1999.     

31P NMR studies of spinach leaves and their chloroplasts

An experimental arrangement is described which enables high quality 31P NMR spectra of compressed spinach leaf pieces to be continuously recorded in which all the resonances observed (cytoplasmic and vacuolar Pi, glycerate-3-P, nucleotides) were sharp and well resolved. 31P NMR spectra obtained from intact chloroplasts showed a distinct peak of stromal Pi. An upfield shift of the stromal Pi resonance was associated with a decrease in the external Pi and vice versa. Nucleotides were largely invisible to NMR in intact chloroplasts, whereas the same nucleotides reappeared in a typical 31P NMR spectrum of an acid extract of intact chloroplasts. Perfusion of compressed spinach leaf pieces with a medium containing Pi triggered a dramatic increase in the vacuolar Pi over 12 h. Addition of choline to the Pi-free perfusate of compressed leaf pieces resulted in a steady accumulation of phosphorylcholine in the cytoplasmic compartment at the expense of cytoplasmic Pi. When a threshold of cytoplasmic Pi concentration was attained, Pi was drawn from the vacuole to sustain choline phosphorylation. In spinach leaves, the vacuole represents a potentially large Pi reservoir, and cycling of Pi through vacuolar influx (energy dependent) and efflux pathways is an efficient system that may provide for control over the cytosolic-free Pi and phosphorylated intermediate concentrations. 31P NMR spectra of neutralized perchloric acid extracts of spinach leaves showed well defined multipeak resonances (quadruplet) of intracellular phytate. The question of cytosolic Pi concentration in green cells is discussed.     

用~（31）P磁共振谱对艾氏腹水癌和肉瘤S－180细胞代谢的研究

艾氏腹水癌细胞和肉瘤Ｓ－１８０细胞是抗肿瘤药物筛选常用细胞株．实验采用取自小鼠腹腔的第７～８天的艾氏腹水癌和Ｓ－１８０细胞,用３１Ｐ磁共振谱测得了细胞内小分子含磷代谢成分;计算了细胞内ｐＨ值;还用３１Ｐ谱探讨了作用机制不同的三种抗代谢物：碘乙酸、２,４－二硝基苯酚及棉酚对艾氏腹水癌细胞代谢的影响     

Identification of phosphorylethanolamine in 31P-NMR spectra of human peripheral blood lymphocytes

The 31P-NMR spectrum of intact human peripheral blood lymphocytes contains a large unidentified peak in the phosphomonoester region. The pH dependency of the 31P-NMR chemical shift of this peak in perchloric acid extracts of peripheral blood lymphocytes was recorded. It was compared to the pH dependency of the chemical shift of phosphorylethanolamine, phosphorylcholine, and ribose 5-phosphate in model solutions. An excellent agreement was found between the behavior of phosphorylethanolamine and the unidentified peak. To further substantiate this assignment phosphorylethanolamine was added to extracts and the pH titrations were repeated. The added phosphorylethanolamine gave exactly the same chemical shift as the unidentified peak and no difference was observed with pH titrations. The concentration of phosphorylethanolamine in human peripheral blood lymphocytes was estimated by 31P NMR to be 2.4 mumol/10(9) cells (range 0.9-4.3/10(9) cells, n = 4).     

Glycinebetaine enhances and stabilizes the evolution of oxygen and the synthesis of ATP by cyanobacterial thylakoid membranes.

Glycinebetaine (betaine), an osmoregulant in halophilic plants, stabilized the evolution of oxygen and the synthesis of ATP by thylakoid membranes from the cyanobacterium Synechocystis PCC6803 when it was present during the preparation and incubation of the thylakoid membranes. Moreover, betaine enhanced the evolution of oxygen and the synthesis of ATP when present during assays. When betaine at 1.0 M was present during the preparation of thylakoid membranes and during the measurement of activity, the rate of evolution of oxygen was equivalent to that of intact cells.     

Measurement of adenosine triphosphate and 2,3-diphosphoglycerate in stored blood with 31P nuclear magnetic resonance spectroscopy

Conditions for blood storage are chosen to assure adequate levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). Because of the invasive nature of the techniques, biochemical assays are not routinely used to measure levels of these compounds in stored blood. However, 31P NMR spectroscopy measures phosphorylated intermediates in intact cells and could be used without disruption of the storage pack. We compared levels of ATP and 2,3-DPG measured by 31P spectroscopy and standard enzyme-linked biochemical assays in whole blood (WB) and packed red blood cells (PRBCs) at weekly intervals during a 35-day storage period. NMR demonstrated a marked decrease in 2,3-DPG and an increase in inorganic phosphate after the first week of storage. No significant differences in ATP concentrations were seen in WB during the storage period, but a significant decrease in ATP in PRBCs was documented. There was good agreement in levels of ATP and 2,3-DPG measured by NMR and biochemical techniques. 31P NMR spectroscopy is a noninvasive technique for measuring ATP and 2,3-DPG which has a potential use in quality assurance of stored blood.     

Rat liver energetics according to data of 31P-NMR in extreme states of the processes of biosynthesis of proteins and nucleic acids

The dynamics of phosphomonoesters, phosphodiesters, Pi, ATP, ADP, NAD(H+) and uridine diphosphoglucose (UDPG) levels in rat liver upon sharp oscillations in the rates of protein and nucleic acid biosynthesis induced by a sublethal++ dose of cycloheximide was studied, using the 31P-NMR method. The results obtained with preparations of native liver are unaffected by fractionation, homogenization and chemical extraction procedures. It was demonstrated that oscillations of Pi, ATP and UDPG levels in liver cells reflect the changes in the energy consumption and intracellular energy-linked processes (e.g., glycolysis, oxidative phosphorylation, glycogen synthesis and consumption) under conditions of variable macromolecular synthesis rates. The oscillations in phosphomonoesters and phosphodiesters levels are mainly due to cycloheximide-induced lipid metabolism disturbances.     

23Na and 31P NMR studies of the effects of salt stress on the freshwater cyanobacterium Synechococcus 6311

We have used 23Na and 31P nuclear magnetic resonance (NMR) spectroscopy to elucidate some of the bioenergetic changes that occur in the freshwater cyanobacterium Synechococcus 6311 after a transition from growth medium (Na concentration 0.01 M) to medium containing 0.5 M NaCl. 23Na NMR analysis showed Na rapidly penetrates the cells under dark aerobic conditions; cells grown for several days in high salt medium, however, reestablish a low internal sodium content, comparable to control cells. For 31P NMR analysis, a system was devised to aerate and illuminate cell suspensions during spectral acquisition. The NMR spectra showed that when cells are presented with 0.5 M NaCl (final concentration), nucleotide triphosphate peaks decrease, the inorganic phosphate peak increases, and the cytoplasmic pH transiently increases from 7.4 to 7.9. Pyrophosphate added to cell suspensions is hydrolyzed to inorganic phosphate apparently by an extracellular phosphatase, allowing external and internal pools of inorganic phosphate to be distinguished. Nucleotide triphosphate levels fall almost as much when cells are incubated in darkness as under anoxia, indicating that both respiration and photosynthesis contribute to the maintenance of intracellular ATP levels. Cells grown in high salt medium for several generations exhibited a pattern of 31P metabolites similar to control cells, except that they produced more (and more intense) peaks in the monoester phosphate region, presumably signals from sugar phosphates.     

31P NMR studies on adenylates and other phosphorus metabolites in the schistosome vector Biomphalaria glabrata

31P nuclear magnetic resonance spectroscopy was applied successfully to whole intact and de-shelled snails and to the isolated digestive gland-gonad tissue complex as well as other tissues of Biomphalaria glabrata. Several phosphorus metabolites, including ATP and ADP, were observed. The mean ATP/ADP ratio calculated for the tissue complex was 3.1 and the ATP concentration was 0.73 nmoles/mg tissue fresh weight. Assignments for AMP, sugar phosphates, and inorganic phosphate peaks were tentatively made. A major phosphorus component was identified as a phosphonate and this metabolite was also present in egg masses and the albumin gland. Phosphoarginine was not observed in the tissue complex but was present in whole animals. Infection by Schistosoma mansoni resulted in marked alteration in the relative levels of phosphorus metabolites in the digestive gland-gonad complex during the course of infection. The decrease in phosphonate was particularly notable. The relative level of a metabolite occurring at -1.1 ppm was also decreased but its identity remained unknown. The ATP/ADP ratio was not affected by infection, but an increase in the relative level of inorganic phosphate suggested a possible decrease in phosphorylation potential.     

31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC----gamma ATP (0.2 mM/s), Pi----gamma ATP (0.2 mM/s) and the conversion beta ATP----beta ADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 mumol/min per mg protein) and adenylate kinase (approx. 0.4 mumol/min per mg protein) suggest that the beta ATP----beta ADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4 X 10(-3) mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.     

Organic phosphate binding to hemoglobin in intact human erythrocytes determined by 31P nuclear magnetic resonance spectroscopy.

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy was used to estimate the percent of 2,3-diphosphoglycerate and ATP bound to hemoglobin in intact human erythrocytes at 37 degrees C. Binding was assessed by comparing the chemical shifts (delta) of 2,3-diphosphoglycerate and of ATP observed in intact cells with the delta values of these organic phosphates determined in model solutions closely simulating intracellular conditions, in which percent binding was directly evaluated by membrane ultrafiltration. The results showed that the percent of bound 2,3-diphosphoglycerate in intact cells varied with pH, the state of oxygenation, and 2,3-diphosphoglycerate concentration. The values ranged from 33% in cells incubated with glucose in air at an intracellular pH of 7.2 to 100% in cells incubated with inosine in N2 at a pH of 6.75. At the same 2,3-diphosphoglycerate concentration, a greater percentage of the compound appeared to be bound in erythrocytes than in the closely simulated model system. ATP was not significantly bound to hemoglobin under any condition examined, but appeared to be strongly complexed to Mg2+ inside the erythrocyte. The binding percentages for both 2,3-diphosphoglycerate and ATP in intact cells estimated by 31P NMR spectroscopy were lower than those calculated by others from individual association constants determined for the binding of different ligands to hemoglobin.     

Interactions of dietary fat and 2,5-anhydro-D-mannitol on energy metabolism in isolated rat hepatocytes

The fructose analog 2,5-anhydro-D-mannitol (2,5-AM) stimulates feeding in rats by reducing ATP content in the liver. These behavioral and metabolic effects occur with rats fed a high-carbohydrate/low-fat (HC/LF) diet, but they are prevented or attenuated when the animals eat high-fat/low-carbohydrate (HF/LC) food. To examine the metabolic bases for this effect of diet, we assessed the actions of 2,5-AM on ATP content, oxygen consumption, and substrate oxidation in isolated hepatocytes from rats fed one of the two diets. Compared with cells from rats fed the HC/LF diet ("HC/LF" cells), cells from rats fed the HF/LC diet ("HF/LC" cells) had similar ATP contents but lower oxygen consumption, decreased fructose, and increased palmitate oxidation. 2,5-AM did not decrease ATP content or oxygen consumption in HF/LC cells as much as it did in HC/LF hepatocytes, and it only affected fructose and palmitate oxidation in HC/LF cells. 31P-NMR spectroscopy indicated that differences in phosphate trapping accounted for differences in depletion of ATP by 2,5-AM. These results suggest that intake of the HF/LC diet prevents the eating response and attenuates the decline in liver ATP by shifting hepatocyte metabolism to favor fat over carbohydrate as an energy-yielding substrate.     

Adenosine Inhibits Choline Kinase Activity and Decreases the Phosphorylation of Choline in Striatal Synaptosomes

The main objective of these studies was to determine whether adenosine inhibits choline kinase in rat striata, leading to a decreased incorporation of choline into phosphorylcholine, a mechanism that may mediate seizure-induced increases in the levels of free choline in brain. Incubation of particulate and soluble fractions of striatal synaptosomes with adenosine or its metabolically stable analogues significantly inhibited enzyme activity. The inhibition was noncompetitive versus choline and competitive versus MgATP. Inhibitor constants for adenosine, 2-chloroadenosine, and 2',5'-dideoxyadenosine at the MgATP site were 94, 49, and 207 microM, respectively; these values were less than the Michaelis constant for MgATP (340 microM). To determine whether adenosine altered the phosphorylation of choline in an intact preparation, synaptosomes were incubated with [3H]choline in the presence or absence of adenosine or its analogues and the amount of [3H]-phosphorylcholine formed from the [3H]choline taken up was measured. All compounds tested significantly reduced the synthesis of [3H]phosphorylcholine. Results suggest that following seizures or hypoxia, when levels of adenosine increase and the concentration of ATP decreases, inhibition of choline phosphorylation may be manifest, resulting in increased levels of free choline in brain.     

Metabolic effects of interleukin 3 on 32D cl23 cells analyzed by NMR

31P NMR of living 32D cl23 cells and 1H NMR of cell extracts were used to study the metabolic effects of interleukin 3 (IL3). When IL3 was removed from 32D cl23 for 9-10 hours 31P spectra showed a decrease in sugar phosphate, gamma ATP/ADP, alpha ATP/ADP/NAD, and beta ATP resonances which declined progressively over a time period of up to 16 hours. By comparison, ATP measurements using the luciferin/luciferase method resulted in the decline of ATP levels from 12 hours in the absence of IL3. At this time, viability of the cells was unaffected. For 1H NMR experiments cells were grown in the presence and absence of IL3 for 4 and 24 hours, after which acid cell extracts were prepared. These spectra revealed a four-fold decrease in lactate 4 hours post-IL3 removal. Alanine levels were unchanged but glycine was elevated 1.5-fold whilst various other amino acids were elevated slightly. After 24 hours without IL3, only 22% of cells were viable which was reflected in a general decline of most resonance intensities. These findings suggest that IL3 exerts its effect primarily on glucose metabolism and has a delayed secondary effect on maintenance of ATP levels in the cell. We have demonstrated the applicability of high resolution 1H and 31P NMR to the study of cellular metabolism in hemopoietic cells.     

Distribution and potential role of cytosolic water-soluble phosphodiesters in fish

The distribution of water-soluble phosphodiesters (WSPDEs) visible by nuclear magnetic resonance (NMR) in some intact tissues of rainbow trout (Oncorhynchus mykiss walbaum) and in perchloric extracts after partial purification was examined by (31)P NMR spectroscopy. The compounds of interest were serine ethanolamine phosphate (SEP), threonine ethanolamine phosphate (TEP), glycerophosphorylcholine (GPC), and glycerophosphorylethanolamine (GPE). TEP and SEP were mostly accumulated in the heart and less accumulated in the kidney of intact trout. After the extraction procedure, two additional minor resonances were visible and identified as GPC and GPE. The liver of trout contained large amounts of GPE. Similar investigations were conducted by (31)P NMR on hearts and kidneys of two elasmobranchs (Scyliorhinus canicula, Raja clavata) and four teleosts (Anguilla anguilla, Sparus auratus, Dicentrarchus labrax, Scophtlhalmus maximus); comparison with the trout data showed striking interspecies differences in the identity of WSPDEs. All teleosts, except eel and turbot, accumulated predominantly TEP. However, in elasmobranchs, first GPC and then GPE were the major compounds. Whatever the studied species, the relative abundances in the heart and kidney were similar. In the last two decades, two hypotheses were proposed to explain the occurrence of high levels of cytosoluble phosphodiesters: these compounds may constitute an index of phospholipid catabolism or a protective mechanism through which phospholipid levels are kept high. To test them and elucidate the role of these compounds in membrane phospholipid regulation in fish, we investigated the effects of two physiological stresses, that is, seawater adaptation and induced myocardial ischemia, on trout cytosolic phosphodiester levels. A 32.5-min ischemic stress caused no effect on SEP and TEP levels. On the contrary, significant osmotic stress induced changes in the PDEs levels: 2 d after transfer from freshwater to seawater or from seawater to freshwater, both tissues displayed a transient decrease of TEP; however, a 2-d stay in seawater after transfer from freshwater caused a rise in SEP concentration, whereas a 2-d stay in freshwater after transfer decreased SEP level. In conclusion, our experiments suggest a relationship between the high levels of cytosoluble phosphodiesters observed in some fish tissues and resistance to stress.     

Choline kinase activity in Plasmodium-infected erythrocytes: characterization and utilization as a parasite-specific marker in malarial fractionation studies

Choline kinase (EC 2.7.1.32) was investigated in plasmodium falciparum-infected erythrocytes. Disrupted infected erythrocytes had a choline kinase activity of 1.9 +/- 0.2 nmol phosphorylcholine/10(7) infected cells per h, whereas the activity in normal uninfected erythrocytes was less than 6 pmol/10(7) cells per h. A broad alkaline optimal pH (7.9-9.2) was observed. The Km values for choline and ATP were 79 +/- 20 microM, and 1.3 +/- 0.3 mM, respectively. ATP concentrations higher than 12 mM inhibited choline kinase. Maximal activity was registered with a Mg2+ concentration of 10 mM, whereas its replacement by Mn2+, or other divalent cations, involved a decrease in choline kinase activity of at least 75%. Inhibition by products of the reaction, such as phosphorylcholine and ADP was investigated. In plasmodium knowlesi-infected erythrocytes, choline kinase had similar properties, but with a much higher specific activity of 16.4 +/- 2.1 nmol/10(7) infected cells per h. Subcellular fractionation of P. knowlesi-infected erythrocyte suspensions revealed that choline kinase was located exclusively in the cytosol of the parasite. We show that this enzyme is a useful index of parasite cytosolic content leakage, when infected erythrocytes are fractionated by saponin lysis or nitrogen decompression.     

Plasma ATP during exercise: possible role in regulation of coronary blood flow

It was previously shown that red blood cells release ATP when blood oxygen tension decreases. ATP acts on microvascular endothelial cells to produce a retrograde conducted vasodilation (presumably via gap junctions) to the upstream arteriole. These observations form the basis for an ATP hypothesis of local metabolic control of coronary blood flow due to vasodilation in microvascular units where myocardial oxygen extraction is high. Dogs (n = 10) were instrumented with catheters in the aorta and coronary sinus, and a flow transducer was placed around the circumflex coronary artery. Arterial and coronary venous plasma ATP concentrations were measured at rest and during three levels of treadmill exercise by using a luciferin-luciferase assay. During exercise, myocardial oxygen consumption increased approximately 3.2-fold, coronary blood flow increased approximately 2.7-fold, and coronary venous oxygen tension decreased from 19 to 12.9 mmHg. Coronary venous plasma ATP concentration increased significantly from 31.1 to 51.2 nM (P < 0.01) during exercise. Coronary blood flow increased linearly with coronary venous ATP concentration (P < 0.01). Coronary venous-arterial plasma ATP concentration difference increased significantly during exercise (P < 0.05). The data support the hypothesis that ATP is one of the factors controlling coronary blood flow during exercise.     

Increased Phosphodiesters in Lipopolysaccharide Prepared from the Polymyxin B-Resistant Isolate of Klebsiella pneumoniae

A polymyxin B (PXB)-resistant mutant of Klebsiella pneumoniae O3 was isolated. Lipopolysaccharide (LPS) extracted from the PXB-resistant isolate bound little PXB, although LPS from the parental strain did. The ³¹P nuclear magnetic resonance (NMR) spectrum of PXB-resistant type LPS showed that it contained much less of the phosphomonoesters and the pyrophosphate esters, and an increased amount of the phosphodiesters, compared to the parental type LPS. The decrease in the binding of PXB might be due to altered phosphate groups on the PXB-resistant type LPS, suggesting that it might explain the PXB-resistance of the mutant.     

The effect of prolonged anoxia at 3°C on tissue high energy phosphates and phosphodiesters in turtles: A 31P-NMR study

Selected tissues (skeletal muscle, heart ventrical, and liver), sampled from turtles (Chrysemys picta bellii) at 3°C either under normoxic conditions or after 12 weeks of anoxic submergence were quantiaatively analysed for intracellular pH and phosphorus metabolites using ³¹P-NMR. Plasma was tested for osmolality and for the concentrations of lactate, calcium, and magnesium to confirm anoxic stress. We hypothesized that, in the anoxic animals, tissue ATP levels would be maintained and that the increased osmolality of the body fluids of anoxic turtles would be accounted for by a corresponding increase in the concentrations of phosphodiesters. The responses observed differed among the three tissues. In muscle, ATP was unchanged by anoxia but phosphocreatine was reduced by 80%; in heart, both ATP and phosphocreatine fell by 35–40%. The reduction in phosphocreatine in heart tissue at 3°C was similar to that observed in isolated, perfused working hearts from turtles maintained at 20°C but no decrease in ATP occurred in the latter tissues. In liver, although analyses of several specimens were confounded by line-broadening, neither ATP nor phosphocreatine was detectable in anoxic samples. Phosphosdiesters were detected in amounts sufficient to account for 30% of normoxic cell osmotic concentration in heart and 11% and 12% in liver and muscle, respectively. The phosphodiester levels did not change in anoxia. Heart ventricular phosphodiester levels in turtles at 3°C were significantly higher than those determined for whole hearts from turtles at 20°C. ¹H, ¹³C and ³¹P NMR analyses of perchloric acid extracts of heart and skeletal muscle from 20°C turtles con firmed that the major phosphodiester observed by NMR in these tissues is serine ethanolamine phosphate. We conclude that the three types of tissues studied differ substantially in their ability to maintain levels of ATP during anoxia, and that liver may continue to function despite NMR-undetectable levels of this metabolite. In addition, we conclude that phosphodiesters do not serve as regulated osmolytes during anoxia, and that the functional significance of their high concentrations in turtle tissues remains uncertain.