In vivo 31P-NMR studies on energy metabolism in and catecholamine effect on rat liver during hypovolemic shock

In vivo 31P-NMR spectroscopy (31P-MRS) was used to study the metabolism of phosphate compounds in rat liver under various conditions. The changes in hepatic concentrations of ATP and inorganic phosphate (Pi) or intracellular pH (pHi) were monitored during hypovolemic shock with or without the infusion of catecholamines. Rapid decreases in the ATP level and pHi with a concomitant increase of Pi were observed upon induction of the hypovolemic shock. Dopamine infusion markedly improved the liver ATP concentration and intracellular acidosis, but epinephrine or norepinephrine were without effects. The present results suggest that dopamine increases abdominal blood flow and improves the energy metabolism in the liver during hypovolemic shock.     

Advantages of perfluorochemical perfusion in the isolated working rabbit heart preparation using 31P-NMR

Quantitative 31P-NMR and enzymatic analysis of high-energy phosphates were used to characterize an isolated perfused working rabbit heart preparation. In this model, the left side of the heart works against a physiological after-load. Two perfusates, Krebs-Henseleit saline and the perfluorocarbon emulsion FC-43 (perfluorotributylamine), were evaluated in their ability to maintain cardiac function and high-energy phosphate metabolites over a period of 2-3 h. Adenine nucleotides ATP, ADP, phosphocreatine and inorganic phosphate (Pi) were measured by 31P-NMR while monitoring cardiac output and coronary flow. Intracellular pH was determined using the chemical shift of Pi. At the end of each experiment, hearts were freeze clamped and enzymatically assayed for adenine nucleotides, phosphocreatine and Pi. In every experiment, hearts perfused with FC-43 emulsion maintained the same rate of cardiac output as hearts perfused with Krebs-Henseleit saline, but with half the coronary flow rate: FC-43, 22 +/- 2.5 (n = 5), Krebs-Henseleit saline 42 +/- 2.7 (n = 6) ml/min, P less than 0.001. Hearts perfused with FC-43 emulsion showed higher [phosphocreatine] and [ATP] measured by 31P-NMR. For [phosphocreatine]: FC-43 3.2 +/- 0.7 (n = 5), Krebs-Henseleit saline 1.7 +/- 0.2 (n = 6) mumol/g wet wt., P less than 0.01. For [ATP]: FC-43 1.8 +/- 0.7 (n = 5), Krebs-Henseleit saline 0.9 +/- 0.2 (n = 6) mumol/g wet wt., P less than 0.02. [phosphocreatine] and [ATP] determined by 31P-NMR values were identical within experimental error to those values obtained by enzymatic analysis. Comparing [Pi] determined by both methods, 36% of Pi in FC-43-perfused hearts, and only 24% of Pi in Krebs-Henseleit saline-perfused hearts were visible by NMR, indicating that a large proportion of Pi is bound in the intact functioning heart. Similar results were obtained for [ADP]. Using the combined techniques of 31P-NMR and enzymatic assay, we have shown in this model of the isolated working rabbit heart preparation, that FC-43 emulsion maintains significantly better function and high-energy phosphate levels than Krebs-Henseleit saline.     

Effect of closantel on intrategumental pH in Schistosoma mansoni and Fasciola hepatica

It has been proposed that the anthelmintic activity of the flukicide, closantel, is due to the drug's ability to interfere with the proton gradient in the parasite's mitochondria that in turn inhibits the generation of ATP by the parasite. Recent results using 31P-NMR suggest that this is not the primary target of the drug. We measured the effects of closantel on fluke intrategumental pH and observed a significant decrease (6.8 to 6.5). This decrease occurred within 10 min and at concentrations that were lower than those that produced significant changes in parasite ATP concentration. We also noted that this drug-induced change in intrategumental pH was associated with a marked reduction in fluke motility. Our results, when coupled to previous reports, would suggest that closantel is a membrane-active molecule that is capable of affecting a number of helminth biochemical and physiological processes.     

A P-NMR study of the intact liver fluke Fasciola hepatica

³¹P-NMR techniques offer a useful method of studying changes in the metabolism of intact parasitic worms. The liver flukes, Fasciola hepatica, provide good quality ³¹P high resolution NMR spectra for at least 6 h under anaerobic conditions. The levels of ATP remain constant throughout this period. There is no signal for phosphocreatine or phosphoarginine. In contrast to the findings in mammalian tissues, there is a distinct peak for the terminal phosphate of ADP. A number of signals are observed in the phosphodiester region of the spectrum the largest of which is identified as l-α-glycerophosphoryl choline. Serotonin (5-hydroxytryptamine) causes an appreciable increase in the levels of sugar phosphates when the flukes are incubated in the absence of glucose. The addition of glucose also causes a marked increase in the signals for the hexose phosphate.     

A 31P-NMR study of bovine adrenocortical mitochondrial metabolic activities

High-field 31P-NMR spectroscopy has been used to study the metabolic activities of coupled bovine adrenocortical mitochondria in vitro. These differentiated organelles use oxygen as a substrate to support both oxidative phosphorylation and specific steroid hydroxylation reactions. The NMR technique allowed the resolution of two inorganic phosphate signals, attributed to the matrix and external medium phosphate pools, at low and high field, respectively. These signals were used to calculate the respective Pi concentrations and to obtain the pH of the two corresponding compartments. In addition, the NMR spectra displayed resonance signals corresponding to ADP added to the medium and to ATP synthesized during oxidative phosphorylation. NMR analysis of the mitochondrial perchloric acid extracts identified the major phosphate-containing metabolites, namely NADP+, NAD+, phosphocholine, phosphoethanolamine, sn-glycero-(3)phosphocholine, AMP, ADP, ATP and Pi. Upon addition of ADP and malate to the oxygenated suspension, the kinetics of mitochondrial external Pi consumption and of ATP synthesis, along with the intra- and extraorganelle pH variations could be monitored over time periods of approximately 30 min, in the absence and presence of different steroid hydroxylation substrates. A major observation was that oxidative phosphorylation, which takes place in the absence of steroid, was markedly inhibited as soon as steroid hydroxylation was operating. These observations show the potential of 31P-NMR spectroscopy in the study of metabolic activities of isolated intact mitochondrial organelles. Such an approach appears promising for further determination of the underlying mechanisms in the balance between vital oxidative phosphorylation and differentiated steroid hydroxylation which are under hormonal control in adrenocortical mitochondria as well as in other steroidogenic cell systems.     

A 31P-NMR study of the intact liver fluke Fasciola hepatica

³¹P-NMR techniques offer a useful method of studying changes in the metabolism of intact parasitic worms. The liver flukes, Fasciola hepatica, provide good quality ³¹P high resolution NMR spectra for at least 6 h under anaerobic conditions. The levels of ATP remain constant throughout this period. There is no signal for phosphocreatine or phosphoarginine. In contrast to the findings in mammalian tissues, there is a distinct peak for the terminal phosphate of ADP. A number of signals are observed in the phosphodiester region of the spectrum the largest of which is identified as l-α-glycerophosphoryl choline. Serotonin (5-hydroxytryptamine) causes an appreciable increase in the levels of sugar phosphates when the flukes are incubated in the absence of glucose. The addition of glucose also causes a marked increase in the signals for the hexose phosphate.     

31P-NMR studies of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis

31P NMR was applied to an examination of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis. Resonances from sugar phosphates, inorganic phosphate, adenylates and arginine phosphate were identified. Two peaks of Pi were identified corresponding to intracellular and extracellular Pi. Anoxia produced an expected decrease in peak intensities of ATP and arginine phosphate while the peak of intracellular Pi was enhanced and shifted to indicate intracellular acidification during anoxia. Spectra of whole larvae were monitored over a temperature range from -30 degrees to +25 degrees C. No abrupt alterations in the spectra were seen at the point of extracellular freezing which occurs at about -8 degrees C but temperature had dramatic effects upon the peak intensities of ATP and arginine phosphate. A reversible increase/decrease in peak intensities, relative to Pi, was observed as temperature was raised/lowered. At 15 degrees and -20 degrees C, the beta peak of ATP was 64% and 2% of the peak intensity of Pi while that of arginine phosphate was 78% and 11%, respectively. This temperature effect was not an artifact of instrumentation (as model solutions containing Pi, ATP and arginine phosphate did not show this effect) or a result of changes in the total amounts of these compounds in the cell with temperature. Rather it is apparent that these molecules become restricted in their rotational movement as temperature is lowered perhaps via binding to subcellular components. Changes in the amounts of freely soluble ATP and arginine phosphate with temperature could have important implications for metabolism and its control. Analysis of the effect of temperature on the chemical shift of Pi was also used to determine pH in the intracellular and extracellular compartments. Temperature change had no effect on extracellular (hemolymph) pH which remained constant at 6.1-6.3. Intracellular pH varied with temperature, however, from pH 6.8 at 15 degrees C to pH 7.3 at -12 degrees C with a change, delta pH/delta 0, of -0.0185 degrees C consistent with alphastat regulation.     

Effects of the anti-cancer drug adriamycin on the energy metabolism of rat heart as measured by in vivo 31P-NMR and implications for adriamycin-induced cardiotoxicity

In vivo 31P-NMR was used to measure the effects of the anti-tumor drug adriamycin on the energy metabolism of rat heart. The exclusive acquisition of NMR signal from cardiac muscle was assured by positioning a solenoidal radio-frequency NMR coil around the heart. Appropriate control experiments verified that 31P-NMR spectra solely originated from this organ. Acute effects occurring shortly after adriamycin administration are expressed in 31P spectra as a dose-dependent decline in the cardiac levels of phosphocreatine, after which stabilization at a new steady-state level occurs. These acute effects of a single dose are complete in 30-60 min and no significant further changes take place within 150 min after drug introduction. Longer-term effects of single high doses and of multiple lower doses were measured up to a week after the initiation of treatment. It seemed that at a total dose of 20 mg/kg, drug-induced interference with cardiac energy metabolism was more pronounced than at the same dose in the acute phase. These 31P-NMR data demonstrate that adriamycin treatment is accompanied by a decrease of the cardiac phosphocreatine/ATP ratio which might be an expression of the well-established cardiotoxicity of the drug.     

Metabolic studies of mammalian cells by 31P-NMR using a continuous perfusion technique

Levels of ATP and Pi in metabolically active Chinese hamster lung fibroblasts were monitored noninvasively by 31P-NMR over many hours and under a variety of conditions. The cells were embedded in a matrix of agarose gel in the form of fine threads which were continuously perfused in a standard NMR tube. The small diameter of the thread allows rapid diffusion of metabolites and drugs into the cells. The changes in ATP and Pi levels were followed as a function of time in response to perfusion with a glucose-containing medium, with isotonic saline and with a medium containing 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. This gel-thread perfusion method should enable routine NMR studies of cellular metabolism, and may have other potential biological applications.     

31P-NMR studies of metabolite compartmentation in Fasciola hepatica

Fasciola hepatica, the common liver fluke, is an anaerobic parasitic worm. Possible compartmentation of metabolites between different cell types, metabolic compartments, and free and macromolecule-bound species was investigated using 31P-NMR. A spectrum of the intact worm shows unusual metabolic features, among which are large amounts of glycerolphosphorylcholine, phospholipids mobile on the NMR time-scale, and free cytosolic ADP. Spectra from cells as different as those in oral sucker tissue and eggs showed similar features. Acidosis after serotonin administration was associated with parallel changes in chemical shifts of intracellular Pi and glucose 6-phosphate, suggesting that they are in the same metabolic compartment. Although 13.4 +/- 1.1 mumol/g wet wt. (n = 3) Mg2+ is present in fluke tissue, a considerable fraction is sequestered out of the cytosol. The intracellular free [Mg2+] was independently estimated from the chemical shifts of ATP and ADP as 1.6 +/- 0.5 mM and 2.9 +/- 0.7 mM, respectively. Quantitation of observable phosphate-containing metabolites in whole tissue and in perchlorate extracts demonstrated that 60% of the total ADP and 50% of the total Pi are 'NMR-invisible' in the intact fluke and therefore probably bound to macromolecules in the cells. The apparent ATP/ADP X Pi free concentration ratio is much lower in this anaerobic tissue than in mammalian oxidative tissues.     

Muscle 31P-NMR in humans: estimate of bias and qualitative assessment of ATPase activity.

A mathematical model is developed whereby the longitudinal magnetization of phosphocreatine (PC), ATP, Pi, and total phosphate (PT) can be calculated on the basis of assumed chemical rate constants (kappa i) and spin lattice relaxation times of the muscle PC in equilibrium ATP in equilibrium Pi exchange system. By means of this model, some unexplained 31P nuclear magnetic resonance (NMR) spectroscopy results from the literature (e.g., a decrease of PT in a closed system) could be explained simply on the basis of the physiological variability of kappa i. Moreover, appropriate model simulations indicate that 1) 31P-NMR spectra obtained with short relaxation delays may be influenced to various extents by the metabolic and physicochemical status of the muscle; 2) the assessment of kappa i by standard NMR spectroscopy techniques may be extremely critical; 3) delta PC/delta Pi, as obtained from conventional 31P-NMR spectra, may represent a sensible index of kappa 2 (the pseudo first-order chemical exchange rate constant of the adenosinetriphosphatase reaction); 4) delta PC/delta Pi changes as detected from sequential (short relaxation delays) 31P-NMR spectra obtained in humans during metabolic transients (e.g., during transition from rest to work and vice versa) may represent an index of transient changes of kappa 2.     

Effects of laser photodynamic therapy on tumor phosphate levels and pH assessed by 31P-NMR spectroscopy

The effects of photodynamic therapy using 632 nm photoradiation emitted from an ion pumped dye laser system on the phosphate metabolite levels of rat mammary tumors were monitored by 31P-NMR spectroscopy. A dramatic decline to almost undetectable levels, in the ratio of whole tumor beta-ATP (NTP) to Pi was observed after systemic administration of 5 mg/kg Photofrin II 24 h prior to exposure of R3230AC rat mammary tumor to laser irradiation at 180 and 360 J/cm2 total fluence. This decline in ATP was accompanied by a concomitant increase in the levels of Pi relative to the total observable phosphate signals. Whole tumor pH was calculated from the chemical shift in inorganic phosphate using the water proton signal as reference. Under the same treatment conditions used to monitor the phosphate metabolites following Photodynamic Therapy, the pH of the tumor as a whole decreased approximately 0.35 units at the time when the beta-ATP to Pi ratios were lowest. This maximal decrease in whole tumor ATP levels and pH, which occurred at 4-6 h post irradiation, was followed by a gradual return to pre-treatment levels over a 24 h period. These results demonstrate that Photodynamic Therapy employing porphyrin photosensitization and monochromatic laser irradiation is effective in reducing both tumor high energy phosphate levels and pH. Depending on sensitizer dose and light fluence, metabolic inhibition, represented by depleted nucleoside triphosphates and elevated Pi, may be reversible.     

Effects of diamfenetide on metabolic and excretory functions of Fasciola hepatica in vitro

The effect of diamfenetide (DFT) on the time course of production of end-products of glucose metabolism, tissue ATP levels and NH3 production by adult Fasciola hepatica in vitro was determined. Products of glucose metabolism are increased between 6 and 24 hr incubation in 10(-4) M DFT. Tissue ATP levels and NH3 production are decreased during this time period. The observed metabolic effects of DFT are manifested at a much later time after drug exposure than previously described membrane-disruptive events indicating that metabolic effects of DFT on F. hepatica may be secondary to the initial effects on surface membranes.     

Evaluation of exercise muscle energetics by NMR

Magnetic resonance imaging (MRI) is superior to ultrasonography and X-CT especially in density resolution in soft tissue. 31P NMR provides information on metabolism, which has not been obtained in vivo by conventional methods, such as phosphocreatine (PCr), inorganic phosphate (Pi), ATP, and intracellular pH. We used MRI and 31P NMR spectroscopy to study skeletal muscle metabolism of human and rat. These NMR results suggested that 1) estimation of muscle fiber composition, 2) evaluation of muscle ATP turnover and 3) imaging of local muscle fatigue are possible.     

The 31P-NMR study of the metabolism of phosphate-containing compounds in the rat liver during inhibition of protein synthesis

Using the 31P-NMR method, the composition of the pool of phosphate-containing metabolites in intact rat liver 72 hours following the blocking of protein biosynthesis by cycloheximide was studied. It was shown that during maximal inhibition, i.e., 2-3 hours after cycloheximide injection, the ATP concentration decreases approximately 5-fold, that of ADP and sugar phosphates--4- and 2-fold, respectively. The intracellular pH in hepatocytes was followed by measuring the chemical shift of the Pi signal. The reconstitution of intracellular pH after 2-3 hours is consistent with changes in the Pi level in hepatocytes. The experimental results were compared with the data of biochemical analysis. NMR seems to be a promising tool in the study of metabolism of various animal organs and tissues under physiological and pathological conditions.     

31P nuclear magnetic resonance of metabolic changes associated with cyanide intoxication in the perfused rat liver.

Metabolic changes associated with cyanide intoxication were observed for the first time in perfused rat liver using ³¹P nuclear magnetic resonance (NMR) at 60.7 MHz. Well-oxygenated control livers showed strong ATP peaks and little discernable internal orthophosphate (Pi). Perfusion with 2 mM cyanide eliminated the observable ATP peaks and caused internal Pi to increase. Despite clear evidence for ATP hydrolysis, resonances from cytoplasmic ADP were conspicuously absent. Resumption of perfusion with cyanide-free buffer caused a dramatic return of the ATP peaks with a concomitant fall in internal Pi. These metabolic changes are consistent with reversible binding of cyanide to mitochondrial cytochromes and their observation by ³¹P NMR indicates the potential of this method for studying metabolism in whole, perfused rat liver under physiologic conditions.     

Phosphorus-31 nuclear magnetic resonance of C6 glioma cells and rat astrocytes. Evidence for a modification of the longitudinal relaxation time of ATP and Pi during glucose starvation

31P-NMR spectroscopy has been used to study the energy metabolism and the NMR visibility of ATP and intracellular Pi of the C6 glioma cell line and rat astrocyte grown on microcarrier beads with the following results. 1. In vivo NMR spectra of C6 glioma cells and rat astrocytes indicate that these cells were able to maintain their level of ATP resonances during a long anoxic period (more than an hour). Both cell types were sensitive to ischemia which induced a loss of ATP resonances within 40 min. Glucose starvation induced by 40% decrease in ATP resonances correlated to a 50% increase in the intensity of the Pi signal. These changes corresponded to a new steady state which could be reversed by reperfusing the cells with a glucose-containing medium. 2. In contrast to in vivo data, 31P-NMR analyses of perchloric acid extracts of cells incubated in a glucose-free medium showed that their ATP and Pi contents were unchanged during starvation. The changes of NMR visibility of the metabolites in living C6 cells were correlated to modifications of their macroscopic longitudinal relaxation times, evolving from 0.30 +/- 0.08 s and 6.6 +/- 1.5 s in the presence of glucose to 0.68 +/- 0.26 s and 3.2 +/- 0.9 s in the absence of glucose for ATP and Pi, respectively. The changes of the NMR detectability of ATP and Pi indicate that changes in their microenvironment occur during glucose starvation, suggesting the existence of different pools of these metabolites within the cells. 3. Under various experimental conditions, i.e. anoxia, ischemia and glucose starvation, rat astrocytes in primary culture showed a very similar behavior to that of C6 cells, suggesting a similar adaptability to the nature of the energy supply for both the normal and the malignant cell.     

Muscle fatigue in McArdle's disease studied by 31P-NMR: effect of glucose infusion

In muscle phosphorylase deficiency (McArdle's disease) there is an abnormally rapid fatigue during strenuous exercise. Increasing substrate availability to working muscle can improve exercise tolerance but the effect on muscle energy metabolism has not been studied. Using phosphorus-31 nuclear magnetic resonance (31P-NMR) we examined forearm muscle ATP, phosphocreatine (PCr), inorganic phosphate (Pi) and pH in a McArdle patient (MP) and two healthy subjects (HS) at rest and during intermittent maximal effort handgrip contractions under control conditions (CC) and during intravenous glucose infusion (GI). Under CC, MP gripped to impending forearm muscle contracture in 130 s with a marked decline in muscle PCr and a dramatic elevation in Pi. During GI, MP exercised easily for greater than 420 s at higher tensions and with attenuated PCr depletion and Pi accumulation. In HS, muscle PCr and Pi changed more modestly and were not affected by GI. In MP and HS, ATP changed little or not at all with exercise. The results suggest that alterations in the levels of muscle PCr and Pi but not ATP are involved in the muscle fatigue in McArdle's disease and the improved exercise performance during glucose infusion.     

31P-NMR and 13C-NMR studies of mannose metabolism in Plesiomonas shigelloides. Toxic effect of mannose on growth.

The metabolism of mannose was examined in resting cells in vivo using 13C-NMR and 31P-NMR spectroscopy, in cell-free extracts in vitro using 31P-NMR spectroscopy, and by enzyme assays. Plesiomonas shigelloides was shown to transport mannose by a phosphoenolpyruvate-dependent phosphotransferase system producing mannose 6-phosphate. However, a toxic effect was observed when P. shigelloides was grown in the presence of mannose. Investigation of mannose metabolism using in vivo 13C NMR showed mannose 6-phosphate accumulation without further metabolism. In contrast, glucose was quickly metabolized under the same conditions to lactate, ethanol, acetate and succinate. Extracts of P. shigelloides exhibited no mannose-6-phosphate isomerase activity whereas the key enzyme of the Embden-Meyerhof pathway (6-phosphofructokinase) was found. This result explains the mannose 6-phosphate accumulation observed in cells grown on mannose. The levels of phosphoenolpyruvate and Pi were estimated by in vivo 31P-NMR spectroscopy. The intracellular concentrations of phosphoenolpyruvate and Pi were relatively constant in both starved cells and mannose-metabolizing cells. In glucose-metabolizing cells, the phosphoenolpyruvate concentration was lower, and about 80% of the Pi was used during the first 10 min. It thus appears that the toxic effect of mannose on growth is not due to energy depletion but probably to a toxic effect of mannose 6-phosphate.     

Chemical assessment of phospholipid and phosphoenergetic metabolites in regenerating rat liver measured by in vivo and in vitro 31P-NMR.

For the assessment of 31P-NMR spectroscopic data, phospholipid precursors (phosphorylethanolamine (PE) and phosphocholine) and catabolites (glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC)), as well as adenosine phosphates were chemically determined in regenerating rat liver. The data were compared with those obtained by in vivo and in vitro 31P-NMR spectroscopies. Chemical assay revealed a significant increase of PE and a decrease of GPE, GPC and ATP in hepatectomy group compared to sham operation group. The values obtained by in vitro NMR were in good agreements with those of chemical assay, but significant differences between the two groups were observed only in PE and inorganic phosphate (Pi). Noticeable increase in PME was not detected by in vivo 31P-NMR spectroscopy, although the increase of PE was about 2.5-times that of the control and its constitution ratio to the whole phosphomonoester (PME) was less than 15%. On the other hand, in vivo NMR showed a large phosphodiester (PDE) peak occupying approx. 40% of the total phosphorus signal, while the contribution of its constituents, GPE and GPC was about 5% found by both chemical assay and in vitro NMR. The PDE peak in in vivo NMR seemed to reflect the membrane phospholipid itself rather than its catabolites. A slight decrease of phosphoenergetic level in regenerating rat-liver was commonly suggested by all three analytical methods.