ATP Resonances Research Articles

Cerebral metabolism after one hyperbaric oxygenation session: 1H and 31P magnetic resonance spectroscopy study

The aim of this study is to elucidate the effect of one HBO session on the concentrations of metabolites detectible by 1H and 31P magnetic resonance spectroscopy. An activation of energy metabolism that manifested in the decrease of creatinephosphate (PCr), pHint and increase α-ATP signal after a HBO session is revealed. Other ATP resonances remain unchanged. Also the reduction of N-acetyl aspartate by 3% in mediolateral prefrontal cortex (MPFC) with p<0.05 and in posterior cingulate cortex (p<0.1) was found. Glutamate levels remained unchanged after HBO session. The demonstrated metabolic changes may signify the activation of power supply processes in order to compensate the energy expenses caused by the excess of O2.

Test-retest analysis of multiple 31 P magnetization exchange pathways using asymmetric adiabatic inversion.

A 31 P-MR inversion transfer (IT) method with a short adiabatic inversion pulse is proposed and its test-retest reliability was evaluated for two spectral fitting strategies. Assessment in a test-retest design (3 Tesla, vastus muscles, 12 healthy volunteers, 14 inversion times, 22 ms asymmetric adiabatic inversion pulse, adiabatic excitation); spectral fitting in Fitting Tool for Interrelated Arrays of Datasets (FitAID) and Java Magnetic Resonance User Interface (jMRUI); least squares solution of the Bloch-McConnell-Solomon matrix formalism including all 14 measured time-points with equal weighting. The cohort averages of k[PCr→γ-ATP] (phosphocreatine, PCr; adenosine triphosphate, ATP) are 0.246 ± 0.050s-1 versus 0.254 ± 0.050s-1 , and k[Pi→γ-ATP] 0.086 ± 0.033s-1 versus 0.066 ± 0.034s-1 (average ± standard deviation, jMRUI versus FitAID). Coefficients of variation of the differences between test and retest are lowest (9.5%) for k[PCr→γ-ATP] fitted in FitAID, larger (15.2%) for the fit in jMRUI, and considerably larger for k[Pi→γ-ATP] fitted in FitAID (43.4%) or jMRUI (47.9%). The beginning of the IT effect can be observed with magnetizations above 92% for noninverted lines while inversion of the ATP resonances is better than -72%. The performance of the asymmetric adiabatic pulse allows an accurate observation of IT effects even in the early phase; the least squares fit of the Bloch-McConnell-Solomon matrix formalism is robust; and the type of spectral fitting can influence the results significantly. Magn Reson Med 78:33-39, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

(31)P-MRS of healthy human brain: ATP synthesis, metabolite concentrations, pH, and T1 relaxation times.

The conventional method for measuring brain ATP synthesis is (31)P saturation transfer (ST), a technique typically dependent on prolonged pre-saturation with γ-ATP. In this study, ATP synthesis rate in resting human brain is evaluated using EBIT (exchange kinetics by band inversion transfer), a technique based on slow recovery of γ-ATP magnetization in the absence of B1 field following co-inversion of PCr and ATP resonances with a short adiabatic pulse. The unidirectional rate constant for the Pi → γ-ATP reaction is 0.21 ± 0.04 s(-1) and the ATP synthesis rate is 9.9 ± 2.1 mmol min(-1) kg(-1) in human brain (n = 12 subjects), consistent with the results by ST. Therefore, EBIT could be a useful alternative to ST in studying brain energy metabolism in normal physiology and under pathological conditions. In addition to ATP synthesis, all detectable (31)P signals are analyzed to determine the brain concentration of phosphorus metabolites, including UDPG at around 10 ppm, a previously reported resonance in liver tissues and now confirmed in human brain. Inversion recovery measurements indicate that UDPG, like its diphosphate analogue NAD, has apparent T1 shorter than that of monophosphates (Pi, PMEs, and PDEs) but longer than that of triphosphate ATP, highlighting the significance of the (31)P-(31)P dipolar mechanism in T1 relaxation of polyphosphates. Another interesting finding is the observation of approximately 40% shorter T1 for intracellular Pi relative to extracellular Pi, attributed to the modulation by the intracellular phosphoryl exchange reaction Pi ↔ γ-ATP. The sufficiently separated intra- and extracellular Pi signals also permit the distinction of pH between intra- and extracellular environments (pH 7.0 versus pH 7.4). In summary, quantitative (31)P MRS in combination with ATP synthesis, pH, and T1 relaxation measurements may offer a promising tool to detect biochemical alterations at early stages of brain dysfunctions and diseases.

Equilibrium Enzymes in Regulatory Systems

See related article, p 68–73 The work by Karamat et al1 contains evidence that creatine kinase (CK) mRNA in vascular smooth muscle has a direct correlation with blood pressure. This result is both interesting and problematic: there is no previous evidence from in vitro experiments that smooth muscle CK is a regulatory enzyme, and there is considerable evidence that the enzyme is at equilibrium inside cells. As the data indicate a regulatory role for the equilibrium enzyme CK in control of blood pressure, the general problem of investigating the role of an equilibrium enzyme in a regulatory system is inherently intriguing. Cytosolic CK has been always considered an equilibrium enzyme, whether in striated or smooth muscle. This assumption was used to calculate the free concentration of ADP in cells, in which the concentrations of ATP, PCr, and creatine could be determined. Phosphate NMR was used to measure the relative free concentrations of ATP, PCr, and Pi, and from the chemical shifts of Pi and β-ATP, the pH and free Mg++ of the cell could also be determined. Coupled with chemical measurements of creatine, and using in vitro measurements of the CK equilibrium constant, the free concentration of ADP (and thus the free energy of the cell) could be calculated. Measurements of free ADP cannot be made from chemical analyses because ADP liberated from f-actin compromises these measurements.2 ADP could not be seen as a peak in striated muscle phosphate NMR because of both its low concentration and chemical shift similarity to the ATP resonances. The lower concentration of Mg++ in smooth muscle meant that β-ADP had a different chemical shift from γ-ATP, and ADP peaks could be seen under …

Energy metabolism and intracellular pH in boar spermatozoa

The effect of energy metabolism on intracellular pH was studied in boar spermatozoa using nuclear magnetic resonance (NMR) spectroscopy and confocal microscopy with the pH-sensitive dye seminaphthorhodafluor (SNARF-1). Freshly ejaculated spermatozoa had a high adenylate energy charge (AEC=0.8), which decreased to 0.6 under aerobic conditions and to 0.2 under anaerobic conditions. Correspondingly, no ATP resonances but high AMP resonance were visible in (31)P-NMR-spectra of the spermatozoa. When an artificial oxygen buffer (Fluosol) and a purpose-built air supply system were used during (31)P-NMR data acquisition, ATP resonances reappeared whereas the AMP resonance disappeared. Boar spermatozoa kept under aerobic conditions have intracellular compartments that differ markedly in pH, as demonstrated by both (31)P-NMR spectroscopy and confocal microscopy. Using confocal microscopy, the midpiece of the flagellum in which all mitochondria are located was identified as an acidic compartment (pH(i-mp) 6.7). The intracellular pH of both the head (pH(i-h)) and the long principal piece of the flagellum (pH(i-pp)) were 7.2 and, thus, only slightly below the extracellular pH (pH(e) 7.3). Storage of spermatozoa in a glucose-free medium at 15 degrees C when they are immotile slowly shifted the pH(i-mp) from 6.7 to 6.9 within 20 h, whereas pH(i-h) and pH(i-pp) remained unchanged (pH 7.1-7.2). When glucose was present in the medium, all visible compartments of the spermatozoa as well as the medium were acidified to pH 6.2 within 20 h. Under these conditions a resonance at 4.8 mg kg(-1) appeared representing glycerol 3-phosphate.

Energy metabolism and intracellular pH in boar spermatozoa.

The effect of energy metabolism on intracellular pH was studied in boar spermatozoa using nuclear magnetic resonance (NMR) spectroscopy and confocal microscopy with the pH-sensitive dye seminaphthorhodafluor (SNARF-1). Freshly ejaculated spermatozoa had a high adenylate energy charge (AEC=0.8), which decreased to 0.6 under aerobic conditions and to 0.2 under anaerobic conditions. Correspondingly, no ATP resonances but high AMP resonance were visible in (31)P-NMR-spectra of the spermatozoa. When an artificial oxygen buffer (Fluosol) and a purpose-built air supply system were used during (31)P-NMR data acquisition, ATP resonances reappeared whereas the AMP resonance disappeared. Boar spermatozoa kept under aerobic conditions have intracellular compartments that differ markedly in pH, as demonstrated by both (31)P-NMR spectroscopy and confocal microscopy. Using confocal microscopy, the midpiece of the flagellum in which all mitochondria are located was identified as an acidic compartment (pH(i-mp) 6.7). The intracellular pH of both the head (pH(i-h)) and the long principal piece of the flagellum (pH(i-pp)) were 7.2 and, thus, only slightly below the extracellular pH (pH(e) 7.3). Storage of spermatozoa in a glucose-free medium at 15 degrees C when they are immotile slowly shifted the pH(i-mp) from 6.7 to 6.9 within 20 h, whereas pH(i-h) and pH(i-pp) remained unchanged (pH 7.1-7.2). When glucose was present in the medium, all visible compartments of the spermatozoa as well as the medium were acidified to pH 6.2 within 20 h. Under these conditions a resonance at 4.8 mg kg(-1) appeared representing glycerol 3-phosphate.

PCr overshoot': a study of the duration in canine myocardium.

The phosphocreatine (PCr) overshoot is a well-documented phenomenon and is readily observable by 31P MRS. In addition, a second 31P MRS observation during ischemia with reperfusion is a diminution in ATP levels. Combining these two as the 'PCr Overshoot' the PCr/ATP ratio may provide an index of viability. However little information is available regarding the duration of this 'overshoot'. For this approach to be useful clinically, the duration of this phenomenon must be ascertained. An open chest canine model of 12 min of ischemia followed by reperfusion (6h) was used. A 2 cm surface coil was sutured to the myocardium and spectra were acquired at 4.7 T. Gated spectra were acquired in <2.5 min with an interpulse delay of 5 s. Integrals of the PCr and ATP (beta) resonances were analyzed using a line-fitting routine. Overall, the PCr signal increased from 22.0+/-0.8 to 25.5+/-0.9 and ATP decreased from 11.7+/-0.4 to 10.0+/-0.4 (arbitrary units). The PCr remained elevated for the entire 6h period and the percentage increase was 15.9%. The ATP remained depleted for the entire 6h period and the percentage decrease was 17.0%. Thus, the clinically relevant and readily observable PCr/ATP is a product of both an increase in PCr and a decrease in ATP for a calculated net increase in PCr/ATP of 39.6%. The PCr/ATP ratio of the ischemia group for baseline, ischemia, 6h reflow, were: 2.33+/-0.18, 1.04+/-0.29 and 3.22+/-0.21. We demonstrate that the 'PCr overshoot' is readily observable and can be monitored noninvasively and nondestructively for 6h. Therefore, the 'PCr overshoot' may be a viable marker of reversible injury in this model and may prove to be applicable for detecting myocardial viability in patients.

Noninvasive measurement of gene expression in skeletal muscle.

We have developed a noninvasive detection method for expression of viral-mediated gene transfer. A recombinant adenovirus was constructed by using the gene for arginine kinase (AK), which is the invertebrate correlate to the vertebrate ATP-buffering enzyme, creatine kinase. Gene expression was noninvasively monitored using (31)P-magnetic resonance spectroscopy ((31)P-MRS). The product of the AK enzyme, phosphoarginine (PArg), served as an MRS-visible reporter of AK expression. The recombinant adenovirus coding for arginine kinase (rAdCMVAK) was injected into the right hindlimbs of neonatal mice. Two weeks after injection of rAdCMVAK, a unique (31)P-MRS resonance was observed. It was observable in all rAdCMVAK injected hindlimbs and was not present in the contralateral control or the vehicle injected limb. PArg and phosphocreatine (PCr) concentrations were calculated to be 11.6 +/- 0.90 and 13.6 +/- 1.1 mM respectively in rAdCMVAK injected limbs. AK activity was demonstrated in vivo by monitoring the decreases in PArg and ATP resonances during prolonged ischemia. After 1 h of ischemia intracellular pH was 6.73 +/- 0.06, PCr/ATP was decreased by 77 +/- 8%, whereas PArg/ATP was decreased by 50 +/- 15% of basal levels. PArg and PCr returned to basal levels within 5 min of the restoration of blood flow. AK activity persisted for at least 8 mo after injection, indicating that adenoviral-mediated gene transfer can produce stable expression for long periods of time. Therefore, the cDNA encoding AK provides a useful reporter gene that allows noninvasive and repeated monitoring of gene expression after viral mediated gene transfer to muscle.

Non-invasive quantitative 31P MRS assay of mitochondrial function in skeletal muscle in situ.

A method for non-invasive, quantitative 31P NMR spectroscopic investigation of mitochondrial function in human skeletal muscle in situ is described. High time resolution 31P NMR measurements of phosphocreatine, inorganic phosphate and ATP resonances were conducted on human forearm flexor muscle during involuntary twitch contraction at eight different frequencies. Mitochondrial and glyco(geno)lytic ATP synthesis fluxes, and the cytosolic free energy of ATP hydrolysis (delta GP), were calculated at incremental steady-states of energy balance. The covariation of mitochondrial ATP synthesis flux, JPMOP, and delta GP was quasi-linear over the physiological range of free energy values. Curve-fit analysis of the covariation yielded a maximal sustainable JPMOP of 0.24 +/- 0.06 mmol ATP l-1.s-1 and a midpoint potential, (delta GP)0.5, of 58.1 +/- 1.2 kJ/mole in the muscle cells.

Application of homonuclear decoupling to measures of diffusion in biological 31P spin echo spectra

Pulsed field gradient (PFG) spin echo 31P NMR can be used to measure diffusion coefficients of phosphorus-containing metabolites in vivo. In biological spin echo spectra, the ATP resonances are phase modulated by J-coupling between the three phosphorus atoms. This phase modulation may severely decrease the apparent signal intensity of the ATP peaks. In this paper, we describe the use of homonuclear decoupling during spin evolution to suppress the effects of J-coupling in biological spin echo spectra. Phosphorous spectra of ATP and creatine phosphate (PCr) in solution and goldfish (Carassius auratus) skeletal muscle demonstrate the effectiveness of homonuclear decoupling in improving the effective signal-to-noise ratio of ATP. In addition, diffusion coefficients of ATP and PCr determined in goldfish skeletal muscle show that PFG homonuclear decoupled spin echo (HDSE) NMR provides accurate measures of diffusion coefficients.

Chemical exchange in tissue extracts revisited: bicarbonate and deuterium isotope effects on 31P resonances of phosphoethanolamine and phosphocreatine.

It is not sufficiently appreciated that chemical exchange can markedly affect the appearance of 31P tissue extract NMR spectra. In addition to the commonly recognized 31P chemical shift effects of divalent metal cation (e.g. Mg2+) binding upon ATP resonances, multiple resonances for phosphoethanolamine (PE) and phosphocreatine (PCr) are observed under certain conditions of pH, temperature, and D2O and bicarbonate concentrations. In the presence of bicarbonate ion (commonly used to neutralize acidic extractions) carbamate formation causes a second 31P resonance for PE to appear. This effect has been described previously for 13C and 1H amino acid resonances in tissue extracts [Sherry et al. J. Magn. Reson. 89, 391-398 (1990)]. The observation of a splitting of the PCr 31P resonance in aqueous solutions containing D2O has been recently ascribed to proton scalar coupling but was described earlier in an underappreciated report [Kupriyanov et al. Biochem. Biophys. Res. Comm. 114, 1117-1125 (1983)] as due to a deuterium isotope effect. These effects, carbamate formation and deuterium isotope shift, are verified herein to cause marked shifts in PE and PCr 31P resonances. The dependence upon experimental parameters is explored.

31P NMR of Mg-ATP in dilute solutions: Complexation and exchange

1. 1. Monovalent-cation [(CH 3) 4N +, K(I), Na(I)] ATP, 1 mM in nucleotide, in aqueous solutions at pH 7.2, 24°C, generates 2 different 31P NMR spectra, depending upon the salt content of the solution. At salt concentrations below 10 mM, the 31P NMR signals are chemically-shifted upfield (Na salt: α, −11.44 δ; β, −22.91 δ; γ, −8.36 δ) and the β- and γ-groups are broadened (at half-height: α, 3.5 Hz; β, 9.6 Hz; γ, 69 Hz). Above 10 mM salt, the signals are shifted downfield and are narrow (Na salt: α, −11.09 δ, 1.9 Hz; β, −21.75 δ, 3.3 Hz; γ, −6.30 δ, 3.9 Hz). 2. 2. The Na-Mg-ATP complex, corresponding to the composition Na 6Mg 1ATP 2, yields a single set of 31P resonances at concentrations of nucleotide of 100 mM, that upon dilution to 0.2 mM, resolve into 2 sets of ATP resonances characterized by low-field and high-field β- and γ-group resonance pairs. This set of ATP resonances, in contrast to the resonance set at 100mM ATP, are broad (100mM in ATP: α, −10.7 δ, 3.7 Hz; β, −20.1 δ, 15 Hz; γ, −5.7 δ, 7.3 Hz. 0.2 mM in ATP: α, −10.7 δ, 47 Hz; β, −18.8 and −21.6 δ, 316 and 274 Hz; y, −5.5 and −8.7 δ, 460 and 374 Hz). 3. 3. This new data, in combination with data derived from a survey of metal-ion-ATP studies, are interpreted in terms of ATP dimers, incorporating 2 molecules of ATP and 2 metal cations, that exist in water under the physiological conditions of neutral pH, high salt content [135mM K(I)] and ATP concentrations in the range of 3 mM. 4. 4. A compilation of 31P in vivo and ex vivo data compared to a reference Mg-ATP chemical shift vs Mg/ATP ratio plot indicates that ATP is not fully Mg-saturated in living systems and that 41% exists as the Mg(ATP); complex.

Avoidance of Hypoxia in a Cnidarian Symbiosis by Algal Photosynthetic Oxygen.

The algal symbionts in a variety of invertebrates are widely believed to increase the oxygen tension in the animal tissue by producing photosynthetic oxygen (1). This could be advantageous to the animal by maintaining normoxia in anoxic waters (2-4). Equally, it could be detrimental, through hyperoxia and the generation of toxic oxygen radicals (5), and this may contribute to the recurrent incidence of mass bleaching in tropical cnidarian symbioses over the last decade (6). Here, we use in vivo 31p nuclear magnetic resonance spectroscopy (NMR) to assess the effect of photosynthetic oxygen production by the symbiotic alga Symbiodinium sp. on the energy metabolism of a sea anemone, Anemonia viridis and, by using acidification of the tissues and elevated ADP/ATP ratios as linked indices of anaerobiosis (7), we show unequivocally that photosynthetic oxygen can protect an invertebrate from hypoxia. Illumination prevents the rapid acidification and reduction in ATP that occurs under hypoxic conditions in the dark, and we suggest that this effect could be partly responsible for algal enhancement of coral calcification. 31p NMR spectra of Anemonia viridis tentacles showed signals from a range of phosphorylated metabolites including phosphonates, orthophosphate (Pi), and ATP (Fig. 1). The spectra were assigned on the basis of an earlier 31p NMR study of sea anemones (8) and from the characteristic chemical shifts of the signals in the spectra. The intensity of the ATP, ADP, and Pi resonances, as well as the position of the Pi signal, were of particular interest here, and the analysis of the spectra was confined to these regions of the spectrum. The chemical shift of the Pi signal

31P MRS of myocardial inorganic phosphate using radiofrequency gradient echoes

Determination of the chemical shift and integral of the myocardial intracellular inorganic phosphate (Pi) resonance by 31P magnetic resonance spectroscopy (MRS) is often precluded due to a large overlapping signal from 2,3-diphosphoglycerate (2,3-DPG) from chamber and myocardial blood. This report demonstrates the use of radiofrequency (RF) magnetic field gradient echoes (RFGE) to eliminate signals from 2,3-DPG in flowing blood, while retaining signals from intracellular myocardial Pi, ATP, and phosphocreatine (PCr). The ECG-triggered 31P spectra were acquired from the myocardium of open chest pigs using a Philips Gyroscan 2-T magnetic resonance spectrometer. A 2.5-cm-diameter surface coil attached to the myocardium was used to provide the RF gradient as well as for excitation and detection of signals. Optimal performance of the RFGE pulse sequence was obtained when the RF gradient pulses were centered at peak diastole or peak systole. Under these conditions, 2,3-DPG signals were completely suppressed, and sensitivity was usually sufficient to allow detection of a well-resolved Pi signal. Myocardial pH determined from RFGE experiments was 7.16 +/- 0.10, and the ratio of the integrals of the Pi and ATP resonances (Pi/ATP) was 0.24. The mean signal-to-noise ratio (S/N) for PCr in control spectra acquired in 4 min was 19/1, while the mean S/N for PCr in RFGE-edited spectra acquired in 15 min was 11/1, demonstrating that the present implementation of the RFGE method results in significant loss in sensitivity. These experiments demonstrate that RFGE-editing allows accurate determination of the chemical shift and integral of the Pi resonance in blood-perfused myocardium in situ.

Phosphorus‐31 nuclear magnetic resonance of C6 glioma cells and rat astrocytes

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.

31P NMR and saturation transfer studies of the effect of Pb2+ on cultured osteoblastic bone cells.

The mechanism of lead toxicity at the cellular level remains unknown, although an effect of lead on intracellular Ca2+ has been described. Since bone is a major target for lead, we have investigated the effect of lead on bioenergetic rates and on the intracellular free Mg2+ concentration in cultured osteoblastic bone cells. Using 31P NMR and the saturation transfer technique we have detected a sizable (18%) transfer of saturation from gamma ATP to Pi in a perfused osteoblastic osteosarcoma bone cell line, Ros 17/2.8, and have found a large (greater than 82%) reduction in the Pi----ATP rate upon treatment with 10 microM Pb2+. The NMR-measured unidirectional rate was much greater than the net rate of ATP synthesis through glycolysis and oxidative phosphorylation. By using iodoacetate we investigated the mechanism of the saturation transfer and found that it is catalyzed by the glycolytic enzyme couple glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. The net rate of glycolysis as measured by lactate production and that of oxidative phosphorylation as measured by O2 consumption were found to be significantly decreased by 18 and 74%, respectively, with lead treatment. In addition, from the chemical shifts of intracellular ATP resonances, we found a significant reduction of 21% in the intracellular free Mg2+ concentration upon Pb2+ treatment. The observed lead-induced reduction in ATP synthesis/utilization and the decrease in intracellular free Mg2+ may contribute to the impairment of bone formation during lead intoxication.

Study of human liver disease with P-31 magnetic resonance spectroscopy.

Liver metabolism and energetics of 24 patients with liver disease were studied using phosphorus-31 magnetic resonance spectroscopy. Significant abnormalities were detected in the majority of these patients. A striking diversity in metabolic patterns was observed. Patients with acute viral hepatitis had low liver phosphodiesters and high phosphomonoesters, possibly phosphocholine and phosphoethanolamine. In alcoholic hepatitis phosphomonoesters were raised. Intracellular inorganic phosphate and inorganic phosphate/ATP ratios were decreased in primary biliary cirrhosis and in some patients with hepatitis. These spectroscopic results were evaluated in respect of the pattern of liver damage and cellular regeneration. Liver tumours had raised phosphomonoesters and also showed evidence for altered spin-lattice relaxation of the phosphorus nucleus in various metabolites. In iron overload the liver ATP resonances were broadened. The line broadening correlated with the degree of iron overload suggesting the potential use of P-31 magnetic resonance spectroscopy for measuring liver iron.

31P nuclear magnetic resonance study of the metabolic pools of adenosine triphosphate in cultured bovine adrenal medullary chromaffin cells.

31P NMR was used to resolve and determine the relative quantity and mobility of ATP in the cytosolic and vesicular compartments of isolated adrenomedullary chromaffin cells. The cells were cultured on microcarrier beads and superfused with an oxygenated medium--thereby permitting dense suspensions of viable cells to be maintained in the NMR probe for extended time periods. Under these conditions, distinct 31P signals could be seen for ATP within the vesicular and the cytosolic pools. Comparison of the integrated areas of the beta-phosphate resonances from the two ATP pools indicated 77% of the endogenous ATP was in the vesicular pool. From this observation and the assumption that the concentration of ATP in the vesicle is 87.5 mM, the concentration of ATP calculated to be in the cytoplasmic pool was approximately 4 mM. The pH in the vesicle determined from the chemical shift of the gamma-phosphate resonance of vesicular ATP was 5.84 +/- 0.17 (n = 6), slightly higher than the intragranular pH measured in hypoxic cells (5.57 +/- 0.15, n = 8). Spin-lattice relaxation times of ATP 31P resonances in the vesicular pool were from 12 to 14 times shorter than the ATP resonances in the cytosol, corresponding to a decrease in molecular mobility due to incorporation of ATP within a catecholamine-storage complex.

A 31P NMR study of the GI tract: effect of fructose loading and measurement of transverse relaxation times.

The effect of fructose loading on high-energy phosphates in the jejunum, ileum, and large intestine of rats was studied using 31P NMR. Following fructose loading, an increase in the intensity of the PME resonance was observed in the jejunum, indicating an accumulation of fructose-1-phosphate. There were no significant changes in ATP or Pi. This demonstrates that the activity of fructokinase in the jejunum can be monitored by 31P NMR. Fructose loading had no detectable effect on metabolite levels in the ileum and large intestine. Resolution of intestinal spectra was poor due to unusually large linewidths and the presence of broad underlying signals. To study the mechanism of line broadening, the T2's of the phosphorus resonances were measured using a solenoidal coil. The T2's of the ATP, Pi, PME, and PCr resonances were much longer than the T2's, suggesting that the linewidths of these resonances are primarily due to susceptibility gradients and/or compartmentation of metabolites. Other signals, particularly in the PDE region, were homogeneously broadened and had very short T2's. Spin echoes obtained with evolution times of 1 to 4 ms suppressed these broad components, with little loss of intensity in the inhomogeneously broadened resonances; as a result, resolution was improved.

Cytoplasmic Mg2+ concentration in platelets: implications for determination of Ca2+ with aequorin.

The concentration of cytoplasmic ionized Mg2+ ([Mg2+]i) varies considerably among different cell types. It has not been measured in platelets. Incorrect estimates of this value could markedly affect many intracellular investigations, including calibration of measurements of platelet cytoplasmic ionized Ca2+ concentration ([Ca2+]i) with the photoprotein aequorin and other Ca2+-sensitive probes. [Mg2+]i was measured in washed, gel-filtered human platelets suspended in modified Tyrode buffer by two methods: 31P-nuclear magnetic resonance (NMR) spectroscopy of intact platelets and null-point titration in platelets selectively permeabilized with digitonin. The 31P-NMR spectra demonstrated that the [Mg2+]i, as calculated from the chemical shift values of ATP resonances, was 0.23 +/- 0.02 (SD) mM in unstimulated platelets. The mean [Mg2+]i as determined by null-point titration was 0.3 +/- 0.1 mM (range: 0.1-0.6 mM). When this [Mg2+]i value was used to construct a Ca2+-calibration curve for aequorin, the indicated [Ca2+]i values in resting and stimulated platelets were lower than those obtained from curves based on previously assumed values for [Mg2+]i (1.0-1.25 mM). This finding largely resolves the discrepancy between resting [Ca2+]i as determined by aequorin or by quin2, fura-2, and indo-1.