In vivo (31)P-NMR diffusion spectroscopy of ATP and phosphocreatine in rat skeletal muscle

The aim of this study was to measure the diffusion of ATP and phosphocreatine (PCr) in intact rat skeletal muscle, using (31)P-NMR. The acquisition of the diffusion-sensitized spectra was optimized in terms of the signal-to-noise ratio for ATP by using a frequency-selective stimulated echo sequence in combination with adiabatic radio-frequency pulses and surface coil signal excitation and reception. Diffusion restriction was studied by measuring the apparent diffusion coefficients of ATP and PCr as a function of the diffusion time. Orientation effects were eliminated by determining the trace of the diffusion tensor. The data were fitted to a cylindrical restriction model to estimate the unbounded diffusion coefficient and the radial dimensions of the restricting compartment. The unbounded diffusion coefficients of ATP and PCr were approximately 90% of their in vitro values at 37 degrees C. The diameters of the cylindrical restriction compartment were approximately 16 and approximately 22 microm for ATP and PCr, respectively. The diameters of rat skeletal muscle fibers are known to range from 60 to 80 microm. The modelling therefore suggests that the in vivo restriction of ATP and PCr diffusion is not imposed by the sarcolemma but by other, intracellular structures with an overall cylindrical orientation.     

In vivo and in vitro 31P-NMR spectroscopy of rat liver treated with halocarbons

Both in vivo and in vitro 31P-NMR spectroscopy were used to demonstrate metabolic changes in rat liver as a function of time after exposure to either carbon tetrachloride (CCl4) or bromotrichloromethane (BrCCl3). The inorganic phosphate resonance, measured in vivo, moves upfield, which is associated with a decrease in cytosolic pH over a 12 or 20 h period (for BrCCl3 or CCl4, respectively). Intoxication by CCl4 or BrCCl3 causes an intracellular acidosis to pH 7.05 or 6.82 (+/- 0.05), respectively. Also, it has been found that halocarbon exposure increases the amounts of phosphomonoesters (PME) detected. High resolution in vitro 31P-NMR spectroscopy studies of perchloric acid extracts of CCl4-treated rat livers indicated a significant increase in the height of the phosphocholine resonance in the PME region 4-5 h after CCl4 exposure.     

Continuous, graded steady-state muscle work in rats studied by in vivo 31P-NMR

Theoretical consideration and experimental findings of 31P nuclear magnetic resonance spectroscopy (NMR) studies of exercising human muscle suggest that a graded, steady-state work protocol is highly suitable for performance evaluation in health and disease. We describe a similar rat model for repeated 31P-NMR studies that follows many of the 31P-NMR features observed in normal human controls. Calf muscles of rats anesthetized with chloral hydrate were indirectly stimulated at four frequencies (0.25, 0.5, 1.0, and 2.0 Hz). It was found that 1) several steady states can be briefly maintained in this model; 2) work-induced phosphocreatine (PCr) fall and inorganic phosphate (Pi) rise is stoichiometric; 3) a linear relationship between stimulation rate and Pi/PCr was obtained, with a slope of 2.01 +/- 0.4 (+/- 2SD, n = 15); 4) no significant drop in ATP was observed, allowing the estimation of phosphorylation potential (PP) changes during this range of muscle work (PP at rest was 61,603 +/- 25,100 M-1 and fell to 6,700 +/- 900 M-1 at the end of exercise); and 5) poststimulation recovery was rapid, with a rate of 2.27 +/- 0.5 PCr/Pi U/min. This simple model can be used for prolonged studies of chronic animal muscle disorders.     

High-energy phosphate metabolism during exercise and recovery in temperate and Antarctic scallops: an in vivo 31P-NMR study

In vivo (31)P-nuclear magnetic resonance (NMR) spectroscopy was used to measure the levels of ATP, phospho-l-arginine (PLA), and inorganic phosphate in the adductor muscle of the Antarctic scallop Adamussium colbecki and two temperate species, Aequipecten opercularis and Pecten maximus. Graded exercise regimes from light (one to two contractions) to exhausting (failing to respond to further stimulation) were imposed on animals of each species at its habitat temperature (0 degrees vs. 12 degrees C, respectively). NMR spectroscopy allowed noninvasive measurement of metabolite levels and intracellular pH at high time resolution (30-120-s intervals) during exercise and throughout the recovery period. Significant differences were shown between the magnitude and form of the metabolic response with increasing levels of exercise in each species. After exhaustion, short-term (first 15 min) muscle alkalosis was followed by acidosis of up to 0.2 pH units during the recovery process. Aequipecten opercularis had similar resting muscle PLA levels compared with either P. maximus or A. colbecki but used a fivefold greater proportion of this store per contraction and was able to perform only half as many claps (maximum of 24) as the other species before exhaustion. All species regenerated their PLA store at a similar rate despite different environmental temperatures. These findings argue for some cold compensation of muscular performance and recovery capacities in the Antarctic scallop, albeit at levels of performance similar to scallops with low activity lifestyles from temperate latitudes.     

The study of energy metabolism in denervated skeletal muscle with 31P-NMR

1. The denervated frog sartorius muscle showed a decrease in the energy store more than that in the control. 2. In the caffeine contractures, both the denervated and the innervated muscles showed similar sequential changes in the relative concentration of phosphocreatine (PCr) to beta-adenosine triphosphate (beta-ATP) and inorganic phosphate (Pi) to beta-ATP. Instead, the intracellular pH value of the denervated muscle was lower than that of the control. 3. It is suggested that phosphate metabolism of the denervated muscle during contracture shows little difference from that of the control, nevertheless, the buffering capacity is decreased in the early stage of atrophy.     

Determination of rat brain buffering in vivo by 31P-NMR

Buffering capacity of most tissues is composed of both rapid and slow phases, the latter presumably due to active acid extrusion. To examine the time course of brain buffering the brain pH of Sprague-Dawley rats was measured using 31P-nuclear magnetic resonance. The effect on brain pH of 30- or 58-min exposures to 20% CO2 followed by 30- or 38-min recovery periods, respectively, was studied. Brain pH reached its lowest value after a 15-min exposure to elevated CO2, thereafter slowly and steadily increasing. During recovery brain pH rose rapidly in the first 5 min exceeding control brain pH by 0.08 pH units. Brain pH fell during the next 30 min despite increases in blood pH and decreases in blood CO2 tension. Calculated intrinsic brain buffering rose steadily threefold during the last 40 min of CO2 exposure and during the final 30 min of recovery. These data show that in rat brain there is a temporally late buffering process, most likely active acid extrusion, requiring greater than 30 min for full activation and at least 30 min for discontinuation.     

In vivo 31P-NMR studies of myocardial high energy phosphate metabolism during anoxia and recovery

31P NMR spectra of heart in-situ in live guinea pigs were obtained continuously in 20.5 s time blocks during 3 min of anoxia, during subsequent reoxygenation and, in separate animals, during terminal anoxia. Reversible anoxia resulted in rapid degradation of phosphocreatine (t 1/2 = 54.5 +/- 2.5 s) which recovered fully during reoxygenation. Heart Pi increased during anoxia and returned to basal levels after oxygen was restored. During 3 min of anoxia, no significant changes in ATP levels or pH were detected. The results demonstrate that it is feasible to measure rapid fluxes of high energy phosphates by 31P NMR in intact animals during and after anoxic stress to the myocardium.     

31P-NMR study of brain phospholipid structures in vivo.

31P-NMR has been used extensively for the study of cytosolic small molecule phosphates in vivo and phospholipid structures in vitro. We present in this paper a series of studies of the brain by 31P-NMR, both in vivo and in extracts, showing the information that can be derived about phospholipids. 31P-NMR spectra of mouse brain at 73 mHz are characterised by almost a complete absence of the large phosphodiester peak in comparison to equivalent spectra at 32 mHz. Proton decoupled spectra in vivo, and spectra of extracts, show that the phosphodiester peak observed in 32 mHz spectra in vivo is mainly due to phospholipid bilayers. Homogenates of quaking and control mouse brains, and of bovine grey matter, show another narrower phosphodiester peak possibly from small phospholipid vesicles. This peak is increased in intensity in the affected mice. These experiments demonstrate the presence of three major components contributing to the phosphodiester resonance: bilayer phospholipids, more mobile phospholipids, and the freely soluble cytosolic molecules glycerophosphocholine and glycerophosphoethanolamine. These NMR methods for non-invasive investigation of phospholipid structures in the brain might be extended to studies of patients with membrane involved diseases such as multiple sclerosis.     

In vivo 31P-NMR studies of Desulfovibrio species. Detection of a novel phosphorus-containing compound

The phosphorus metabolism of sulfate-reducing bacteria was, for the first time, probed by in vivo 31P NMR. A novel phosphoric anhydride diester compound was detected in Desulfovibrio desulfuricans ATCC 27774 at intracellular concentrations up to 5 mM. The compound has been extracted and partially purified by anion-exchange chromatography and analysed by 31P, 13C and 1H NMR. These studies show that the novel phosphorus-containing compound is formed by five carbon atoms and is probably cyclic, with a Mr of approximately 300. Various Desulfovibrio strains were examined in vivo for the presence of this phosphorus-containing compound. Detectable amounts of the novel metabolite were found in D. desulfuricans ATCC 27774 when grown on lactate/sulfate, lactate/thiosulfate or pyruvate/sulfate. The phosphorus-containing compound was not detected when this strain of D. desulfuricans was grown on lactate/nitrate or pyruvate; neither was it detected in two other strains which, like D. desulfuricans ATCC 27774, have the capability of utilizing nitrate as a terminal electron acceptor.     

Forearm metabolic asymmetry detected by 31P-NMR during submaximal exercise

This study evaluated the relationship of skeletal muscle energy metabolism to forearm blood flow and muscle mass in the dominant (D) and nondominant (ND) forearms of normal subjects. 31P-Magnetic resonance spectroscopy was used to determine intracellular pH and the ratio of inorganic phosphate to phosphocreatine (Pi/PCr), an index of energy metabolism. Forearm blood flow and muscle mass were measured by venous occlusion plethysmography and magnetic resonance imaging, respectively. Metabolic measurements and flow were determined at rest and during submaximal exercise in both forearms. After a warm-up period, six normal right-handed male subjects performed 7.5 min of wrist flexion exercise in the magnet (1 contraction every 5 s), first with the ND forearm and then with the D forearm, at 23, 46, and 69 J/min. At rest, there were no differences between forearms in Pi/PCr or pH. However, at each work load the D forearm demonstrated significantly lower Pi/PCr and higher pH than the ND forearm. Blood flow was not significantly different between the forearms at rest or during exercise. Because these subjects were not engaged in unilateral arm training, we conclude that 1) Pi/PCr is lower and pH is higher in the D compared with the ND forearm in normal subjects during submaximal exercise, 2) these differences are independent of muscle mass and blood flow, and 3) the cumulative effect of long-term, low-level daily activity provides an adequate training stimulus for muscular metabolic adaptations.     

Microviscosity of human erythrocytes studied with hypophosphite and 31P-NMR

A 31P-NMR method, which complements earlier 13C-NMR procedures for probing the intra-erythrocyte microenvironment, is described. Hypophosphite is an almost unique probe of the erythrocyte microenvironment, since it is rapidly transported into the cell via the band 3 protein, and intra- and extracellular populations give rise to distinct resonances in the 31P-NMR spectrum. Relaxation mechanisms of the 31P nucleus in the hypophosphite ion were shown to be spin-rotation and dipole-dipole. Analysis of longitudinal relaxation rates in human erythrocytes, haemolysates and concentrated glycerol solutions allowed the determination of microviscosity using the Debye equation. Bulk viscosities of lysates and glycerol solutions were measured using Ostwald capillary viscometry. Translational diffusion coefficients were then calculated from the viscosity estimates using the Stokes-Einstein equation. The results with a range of solvent systems showed that 'viscosity' is a relative phenomenon and that bulk (i.e., macro-) viscosity is therefore not necessarily related to the NMR-determined viscosity. The intracellular NMR-determined viscosities from red cells, ranging in volume from 65.5 to 100.1 fl, varied from 2.10 to 2.67 mPa s. This is consistent with the translational diffusion coefficients of the hypophosphite ion altering by only 20%, whereas the values determined from bulk viscosity measurements conducted on lysates of these cells are consistent with a 230% change.     

31P-NMR saturation transfer study of the in vivo kinetics of arginine kinase in Carcinus crab leg muscle

The kinetics of the reaction catalyzed by arginine kinase have been determined at 9.5 and 23°C for in vivo leg muscle of Carcinus maenas (the common shore crab) using the noninvasive technique of ³¹P-NMR spectroscopy. Concentrations of mobile phosphorus metabolites were the same at both temperatures: 78.7 mM for arginine phosphate, 9.0 mM for adenosine triphosphate (ATP), and 2.6 mM for inorganic phosphate (P_i), as estimated from NMR resonance intensities and literature values for ATP concentration as assayed by traditional biochemical methods. Apparent unidirectional rate constants for formation of ATP from arginine phosphate and ADP were 0.09 s^?1 at 9.5°C and 0.27 s^?1 at 23°C. Pseudo-first-order rate constants for arginine phosphate generation from Arg and ATP were 0.38 and 1.10 s^?1 at 9.5 and 23°C, respectively. In vivo Q₁₀ for the arginine kinase reaction between 9.5 and 23°C was thus 2.2 for both directions. When the kinetic data are analyzed using the Arrhenius equation, activation energies of 126 kJ/mol for ATP formation and 105 kJ/mol for arginine phosphate formation are found. The measured chemical fluxes through arginine kinase in the forward reaction (arginine phosphate hydrolysis) were twice those in the reverse reaction, consistent with either compartmentation of substrates or participation of substrates in alternative metabolic pathways.     

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.     

31P-NMR study of the regulation of glycogenolysis in living skeletal muscle

Based on in vitro biochemical experiments, it is generally believed that glycogenolysis is regulated in two different ways; i.e., Ca2+ regulation at the phosphorylase step and phosphate-product regulation at the phosphofructokinase step. Recent studies on glycogenolysis in living vertebrate skeletal muscles using 31P nuclear magnetic resonance (NMR) presented evidence that glycogenolysis in vivo is regulated by Ca2+ released from the sarcoplasmic reticulum. We performed 31P-NMR studies on living frog skeletal muscle, and found that glycogenolysis is further regulated by the accumulation of phosphate products by contractile activity. Therefore, glycogenolysis in vivo can actually be regulated by the two mechanisms as predicted by in vitro biochemical studies.     

31P-NMR spectroscopy and the metabolic properties of different muscle fibers

To study the in vivo recruitment of different fiber types and their metabolic properties, 31P-nuclear magnetic resonance spectroscopy (31P-NMRS) of the human calf muscle was performed in seven normal sedentary subjects. In the exhaustive exercise protocol used, the work load was increased every minute during 5 min. This resulted in a prominent split of the Pi resonance in all subjects, indicating pH compartmentation in the muscles studied. From the chemical shift of the Pi peaks relative to phosphocreatine (PCr) at the end of the exercise, intracellular pH (pHi) averaged 6.92 +/- 0.05 (SD) in compartment 1 and 6.23 +/- 0.15 in compartment 2. The recovery of both Pi resonances after exercise could be followed easily in five of these subjects. The recovery rate of the Pi peak is a good estimate of the oxidative metabolism at the end of the exercise. A monoexponential regression analysis showed that the mean initial recovery rate S0 was 2.49 +/- 0.17%/s in compartment 1 and only 0.87 +/- 0.12%/s in compartment 2, indicating aerobic function three times higher in compartment 1 at the end of exercise. The mean relative ATP fraction dropped significantly (P less than 0.001), from 20.0 +/- 1.0% of the total 31P signal integral before exercise to 14.0 +/- 1.6% at the end of exercise. The simultaneous visualization of two compartments, in good order, one with high pHi and fast recovery and another with low pHi and slow recovery, is rationalized by the different metabolic behavior of type I and II fibers in human calf muscle in response to exhaustive exercise. This study demonstrates that 31P-NMRS is an excellent noninvasive procedure to quantify aerobic metabolism in both fiber types simultaneously.