Cerebral metabolism in brain tumor of mice studied by in vivo 31P-NMR spectroscopy

We now report a mouse model system of brain tumor for 31P-NMR spectroscopic study of in vivo cerebral metabolism. In vivo 31P-NMR (109 MHz) spectra were taken on the 9th day by the Faraday shield method of the brain of mice (3-week-old) transplanted intracerebrally with mKS X A tumor cells. In tumor-bearing mice, the amount of creatine phosphate decreased markedly and that of inorganic phosphate plus sugar phosphate increased accordingly. Furthermore, the broadening and splitting of individual signals were also noted with tumor-bearing mice; this is interpreted as indicating a variety of changes in chemical shift occurring in the brain of the animals due to heterogeneous distribution of pH. Binding or detaching of divalent cations to and from phosphometabolites may also be responsible for these changes.     

pH heterogeneity in tibial anterior muscle during isometric activity studied by (31)P-NMR spectroscopy.

The occurrence of pH heterogeneity in human tibial anterior muscle during sustained isometric exercise is demonstrated by applying (31)P-nuclear magnetic resonance (NMR) spectroscopy in a study of seven healthy subjects. Exercise was performed at 30 and 60% of maximal voluntary contraction (MVC) until fatigue. The NMR spectra, as localized by a surface coil and improved by proton irradiation, were obtained at a high time resolution (16 s). They revealed the simultaneous presence of two pH pools during most experiments. Maximum difference in the two pH levels during exercise was 0.40 +/- 0.07 (30% MVC, n = 7) and 0.41 +/- 0.03 (60% MVC, n = 3). Complementary two-dimensional (31)P spectroscopic imaging experiments in one subject supported the supposition that the distinct pH pools reflect the metabolic status of the main muscle fiber types. The relative size of the P(i) peak in the spectrum attributed to the type II fiber pool increases with decreasing pH levels. This phenomenon is discussed in the context of the size principle stating that the smaller (type I) motor units are recruited first.     

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.     

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.     

Phosphorus metabolism and intracellular pH in the halotolerant alga Dunaliella parva studied by 31P-NMR

The metabolism of the green unicellular halotolerant alga Dunaliella parva was studied by means of ³¹P nuclear magnetic resonance spectroscopy. The major soluble phosphate compounds were found to be similar to those in other organisms but two phosphodiesters, glycerophosphorylglycerol and glycerophosphorylcholine, were identified in algal tissue for the first time. Only a single pool of intracellular orthophosphate was observed and the chemical shift of the corresponding resonance was used to monitor the intracellular pH. The cell pH and the orthophosphate content were sensitive both to the oxygenation of the cells and to the illumination of the cell suspension. The intracellular pH was controlled over an external pH range of 6–9, but at pH 5 the cell contents became acidic. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone was observed to uncouple oxidative phosphorylation but it did not equilibrate the pH difference across the cell membrane in experiments conducted at an external pH of 7.8.     

Changes in intracellular pH and inorganic phosphate concentration during and after muscle contraction as studied by time-resolved 31P-NMR. Alkalinization by contraction

The morula and the mesenchyme blastula nuclei contained approx. 30 nuclear proteins which were preferentially released by limited digestion with DNase I, but no proteins were released from sperm nuclei. While most of the proteins released by DNase I digestion were common to the two embryonic stages, 2 and 6 proteins were specific or enriched in morulae and mesenchyme blastulae, respectively.     

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.     

Regulation of intracellular pH in LLC-PK1 cells studied using 31P-NMR spectroscopy

31P-NMR spectroscopy was used to monitor intracellular pH (pHi) in a suspension of LLC-PK1 cells, a renal epithelial cell line. The regulation of intracellular pH (pHi) was studied during intracellular acidification with 20% CO2 or intracellular alkalinization with 30 mM NH4Cl. The steady-state pHi in bicarbonate-containing Ringer's solution (pHo 7.40) was 7.14 +/- 0.04 and in bicarbonate-free Ringer's solution (pHo 7.40) 7.24 +/- 0.04. When pHo was altered in nominally HCO3(-)-free Ringer's, the intracellular pHi changed to only a small extent between pHo 6.6 and pHo 7.6; beyond this range pHi was linearly related to pHo. Below pHo 6.6 the cell was capable of maintaining a delta pH of 0.2 pH unit (inside more alkaline), above pH 7.6 a delta pH of 0.4 unit could be generated (inside more acid). During exposure to 20% CO2 in HCO3(-)-free Ringer's solution, pHi dropped initially to 6.9 +/- 0.05, the rate of realkalinisation was found to be 0.071 pH unit X min-1. After removal of CO2 the pHi increased by 0.65 and the rate of reacidification was 0.056 pH unit X min-1. Exposure to 30 mM NH4Cl caused a raise of pHi by 0.48 pH unit and an initial rate of re-acidification of 0.063 pH unit X min-1, after removal of NH4Cl the pHi fell by 0.58 pH unit below the steady-state pHi, followed by a subsequent re-alkalinization of 0.083 pH unit X min-1. Under both experimental conditions, the pHi recovery after an intracellular acidification, introduced by exposure to 20% CO2 and by removal of NH4+, was found to be inhibited by 53% and 63%, respectively, in the absence of sodium and 60% and 72%, respectively, by 1 mM amiloride. These studies indicate that 31P-NMR can be used to monitor steady-state intracellular pH as well a pHi transients in suspensions of epithelial cells. The results support the view that LLC-PK1 cells use an Na+-H+ exchange system to readjust their internal pH after acid loading of the cell.     

Relationship between intracellular pH and energy metabolism in dog brain as measured by 31P-NMR

The relationships between pHi (intracellular pH) and phosphate compounds were evaluated by nuclear magnetic resonance (NMR) in normo-, hypo-, and hypercapnia, obtained by changing fractional inspired concentration of CO2 in dogs anesthetized with 0.75% isoflurane and 66% N2O. Phosphocreatine (PCr) fell by 2.02 mM and Pi (inorganic phosphate) rose by 1.92 mM due to pHi shift from 7.10 to 6.83 during hypercapnia. The stoichiometric coefficient was 1.05 (r2 = 0.78) on log PCr/Cr against pHi, showing minimum change of ADP/ATP and equilibrium of creatine kinase in the pH range of 6.7 to 7.25. [ADP] varied from 21.6 +/- 4.1 microM in control (pHi = 7.10) to 26.8 +/- 6.3 microM in hypercapnia (pHi = 6.83) and 24.0 +/- 6.8 microM in hypocapnia (pHi = 7.17). ATP/ADP X Pi decreased from 66.4 +/- 17.1 mM-1 during normocapnia to 25.8 +/- 6.3 mM-1 in hypercapnia. The ADP values are near the in vitro Km; thus ADP is the main controller. The velocity of oxidative metabolism (V) in relation to its maximum (Vmax) as calculated by a steady-state Michaelis-Menten formulation is approximately 50% in normocapnia. In acidosis (pH 6.7) and alkalosis (pH 7.25), V/Vmax is 10% higher than the normocapnic brain. This increase of V/Vmax is required to maintain cellular homeostasis of energy metabolism in the face of either inhibition at extremes of pH or higher ATPase activity.     

Changes in phosphometabolites and intracellular pH in the tail muscle of the prawnPalaemon serratus as shown by in vivo31P-NMR

Summary ³¹P NMR spectra were recorded from tail muscles of the prawnPalaemon serratus, at rest, after exhaustive work and during subsequent recovery. At rest, the spectra indicated concentrations of phosphoarginine and ATP in good agreement with those obtained from resting fast skeletal muscles in mammals, which are characterized by a high phosphocreatine/P_i ratio. Following exhaustive work, phosphoarginine dropped by ca. 60% and ATP by 20%, while inorganic phosphate increased by 160%. The increase in inorganic phosphate immediately after contractions and in the first minutes of recovery corresponded partially to the changes in phosphoarginine and ATP. During recovery, the decrease of inorganic phosphate balanced the resynthesized phosphoarginine which was fully replenished within 30–40 min. The position of the inorganic phosphate resonance peak was used to monitor changes in intracellular pH (pH_i). The average pH_i in resting tail muscles was 7.20. After stimulation it was observed to decrease by 0.22 units. The return to pre-stimulation value was not achieved within 45 min. A NMR index (ATP+Arg-P)/(ATP+Arg-P+P_i) was calculated to characterize the extent of energetic changes caused by exercise.     

Hairpins in a DNA site for topoisomerase II studied by 1H- and 31P-NMR.

1H- and 31P-NMR and UV-absorption studies were carried out with the oligonucleotide strands d(AGCT-TATC-ATC-GATAAGCT) (-ATC-) and d(AGCTTATC-GAT-GATAAGCT) (-GAT-) contained in the strongest and salt resistant cleavage site for topoisomerase II in pBR322 DNA. We found that the two oligonucleotides were stabilized under a hairpin structure characterized by a eight base pair stem and a three base loop at low DNA and salt concentrations. In such experimental conditions, only the -GAT- oligonucleotide displayed a partial homoduplex structure in slow equilibrium with its folded structure. Temperature dependencies of imino protons showed that the partial homoduplex of -GAT- melted at a lower temperature than the hairpin structure. It was suggested that the appearance of the partial homoduplex in -GAT- is related to the formation of two stabilizing (G.T) mismatched base pairs in the central loop of this structure. Finally, it was inferred from the dispersion of chemical shifts in the 31P-NMR spectra that the distortions affecting the backbone of the hairpin loop are larger in the case of -ATC- compared with -GAT-. At the same time NOEs proved that the base stacking was stronger within the loop of the -ATC- hairpin.     

Temperature-dependent lateral diffusion of phospholipids in hepatic microsomes as studied by 31P-NMR

The 81 MHz 31P-NMR spectra of isolated rabbit liver microsomes before and after trypsin treatment and of the total microsomal lipid extract were recorded in the 4-40 degrees C temperature range. In both treated and untreated microsomes at 4 degrees C most of the phospholipids gave rise to typical bilayer spectra whereas the lineshape of the latter in the 25-37 degrees C temperature range becomes narrower and more symmetrical. Quasi-elastic light scattering (QELS) measurements revealed that the microsomes maintain their size in the temperature region of the measurements. We interpret the lineshape changes for untreated microsomes above 25 degrees C as being determined by lateral diffusion. This is supported by lineshape calculations as a function of the lateral diffusion coefficient. The different spectral behavior of enzymatically active (untreated) and inactive (treated) microsomes suggests that the membrane proteins influence the lateral diffusion of the phospholipids.     

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.     

Changes in Cytoplasmic pH Measured by 31P-NMR in Cells of Nitellopsis obtusa

The cytoplasmic pH of Nitellopsis obtusa measured by the ³¹P-nuclearmagnetic resonance method was about 7.3. Since normal cell havea large vacuole, a long period of time is needed to accumulatesignals for cytoplasmic phosphate. To measure the cytoplasmicpH in a shorter period, cytoplasm-rich cells were prepared bycentrifugation. The cytoplasmic pH of these cells was also about7.3. Illumination reversibly increased the cytoplasmic pH from7.3 to 7.8. When the cytoplasm-rich cell was illuminated, themembrane potential hyperpolarized concomitant with the alkalificationof the cytoplasm. (Received December 5, 1983; Accepted May 12, 1984)     

Structural transitions in short-chain lipid assemblies studied by (31)P-NMR spectroscopy

The self-assembled supramolecular structures of diacylphosphatidylcholine (diC(n)PC), diacylphosphatidylethanolamine (diC(n)PE), diacylphosphatidyglycerol (diC(n)PG), and diacylphosphatidylserine (diC(n)PS) were investigated by (31)P nuclear magnetic resonance (NMR) spectroscopy as a function of the hydrophobic acyl chain length. Short-chain homologs of these lipids formed micelles, and longer-chain homologs formed bilayers. The shortest acyl chain lengths that supported bilayer structures depended on the headgroup of the lipids. They increased in the order PE (C(6)) < PC (C(9)) < or = PS (C(9) or C(10)) < PG (C(11) or C(12)). This order correlated with the effective headgroup area, which is a function of the physical size, charge, hydration, and hydrogen-bonding capacity of the four headgroups. Electrostatic screening of the headgroup charge with NaCl reduced the effective headgroup area of PS and PG and thereby decreased the micelle-to-bilayer transition of these lipid classes to shorter chain lengths. The experimentally determined supramolecular structures were compared to the assembly states predicted by packing constraints that were calculated from the hydrocarbon-chain volume and effective headgroup area of each lipid. The model accurately predicted the chain-length threshold for bilayer formation if the relative displacement of the acyl chains of the phospholipid were taken into account. The model also predicted cylindrical rather than spherical micelles for all four diacylphospholipid classes and the (31)P-NMR spectra provided evidence for a tubular network that appeared as an intermediate phase at the micelle-to-bilayer transition. The free energy of micellization per methylene group was independent of the structure of the supramolecular assembly, but was -0.95 kJ/mol (-0.23 kcal/mol) for the PGs compared to -2.5 kJ/mol (-0.60 kcal/mol) for the PCs. The integral membrane protein OmpA did not change the bilayer structure of thin (diC(10)PC) bilayers.     

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.