Phosphorus magnetic resonance spectroscopy: its utility in examining the membrane hypothesis of schizophrenia

A novel approach to understanding the pathophysiology of schizophrenia has been the investigation of membrane composition and functional perturbations, referred to as the "Membrane Hypothesis of Schizophrenia." The evidence in support of this hypothesis has been accumulating in findings in patients with schizophrenia of reductions in phospholipids and essential fatty acids various peripheral tissues. Postmortem studies indicate similar reductions in essential fatty acids in the brain. However, the use of magnetic resonance spectroscopy (MRS) has provided an opportunity to examine aspects of membrane biochemistry in vivo in the living brain. MRS is a powerful, albeit complex, noninvasive quantitative imaging tool that offers several advantages over other methods of in vivo biochemical investigations. It has been used extensively in investigating brain biochemistry in schizophrenia. Phosphorus MRS (31P MRS) can provide important information about neuronal membranes, such as levels of phosphomonoesters that reflect the building blocks of neuronal membranes and phosphodiesters that reflect breakdown products. 31P MRS can also provide information about bioenergetics. Studies in patients with chronic schizophrenia as well as at first episode prior to treatment show a variety of alterations in neuronal membrane biochemistry, supportive of the membrane hypothesis of schizophrenia. Below, we will briefly review the principles underlying 31P MRS and findings to date. Magnetic resonance spectroscopy (MRS) is a powerful, albeit complex, imaging tool that permits investigation of brain biochemistry in vivo. It utilizes the magnetic resonance imaging hardware. It offers several advantages over other methods of in vivo biochemical investigations. MRS is noninvasive, there is no radiation exposure, does not require the use of tracer ligands or contrast media. Because of it is relatively benign, repeated measures are possible. It has been used extensively in investigating brain biochemistry in schizophrenia.     

Reactivity of Al(III) with membrane phospholipids: a NMR approach

The complexes Al(acac)₃ (1) (acac = 2,4-pentanedionate) and Al(malt)₃ (malt = 3-hydroxy-2-methyl-4-pyronate) (2) react with dl--dipalmitoylphosphatidylcholine (DPPC) under a 1:1 molar ratio in CDCl₃ at 37 °C, as shown by the substantial release of ligands (20–50%) from the metal coordination sphere (¹H-NMR), by evident changes in the ¹H-NMR spectrum of DPPC in the reaction mixture and by the appearance of a ³¹P-NMR signal due to metal-coordinated DPPC. ³¹P-NMR spectra reveal that both 1 and 2 also react with DPPC in water, in the presence of 1% Triton X-100 and Tris buffer. Under these conditions, 1 and 2 do not react with ghosts from human erythrocytes. On the contrary, the far less hydrolytically stable complex Al(lact)₃ (lact = lactate) appears to be reactive under identical conditions, as shown by ³¹P-NMR spectra.     

Cerebral metabolism in male patients with schizophrenia who have seriously and dangerously violently offended: a 31P magnetic resonance spectroscopy study

There is biochemical evidence to suggest that membrane phospholipid metabolism may be impaired in some patients with schizophrenia. The aim of this study was to test the hypothesis that patients with schizophrenia who have violently offended while psychotic suffer from changes in cerebral phospholipid metabolism. Cerebral 31-phosphorus magnetic resonance spectroscopy was carried out in 15 male patients with schizophrenia who had violently offended (homicide, attempted murder, or wounding with intent to cause grievous bodily harm) while psychotic and in a control group of 13 age-matched healthy male control subjects. Spectra were obtained from 70x70x70mm(3) voxels in the brain using an image-selected in vivo spectroscopy pulse sequence. betaNTP was lower (P < 0.04) and gammaNTP was higher (P < 0.04) in the patient group compared with the normal control group. Our results are suggestive of increased cerebral energy metabolism taking place in the forensic patients.     

Brain development:1H magnetic resonance spectroscopy of rat brain extracts compared with chromatographic methods

We compared in vitro¹H magnetic resonance spectroscopy (MRS) measurements of rat brain extracts (rats: 2–56 days old) with chromatographic measurements and in a further step also with results of in vivo MRS. The following substances can be reliably measured in brain extracts by in vitro MRS: N-acetylaspartate (NAA), total creatine (Cr), phosphorylethanoloamine (PE), taurine (Tau), glutamate (Glu), glutamine (Gln), -aminobutyrate (GABA) and alanine (Ala). Two different methods of MRS data evaluation compared with chromatographic data on Cr and NAA are shown. During development of the rat from day 2–56 brain concentrations of PE, Tau and Ala decrease, those of NAA, Cr, Glu and Gln increase, while GABA does not change. The developmental patterns of these substances are the same, whether measured by in vitro MRS or by chromatographic methods. Quantification of NAA, Cr, Tau, GABA and PE leads to the same results with both methods, while Glu, Gln and Ala concentrations determined by in vitro MRS are apparently lower than those measured chemically. The NAA/Cr ratios of 7 to 35-day-old rats were determined by in vivo¹H MRS. These results correlate with chromatographic and in vitro data. Using appropriate methods in the in vivo and in vitro MR-technique, the obtained data compare well with the chromatographic results.     

Head-group conformation in phospholipids: a phosphorus-31 nuclear magnetic resonance study of oriented monodomain dipalmitoylphosphatidylcholine bilayers

Angular-dependent 31P NMR spectra of oriented biaxial monodomain DPPC.H2O multilayers are employed to study head-group conformation in this phospholipid. The results indicate that the O-P-O plane of the phosphate, where the O's are the nonesterified oxygens of the phosphodiester, is tilted at 47 +/- 5 degrees with respect to the bilayer normal. This PO4 orientation could result in the choline moiety being extended parallel to the bilayer plane, and it will explain the breadth of the axially symmetric 31P powder spectrum observed for DPPC in excess water. This work is the first direct observation of this conformation for lecithins and it illustrates the utility of high-resolution solid-state NMR in structural studies of disordered systems.     

Negative correlation between cerebral inorganic phosphate and the volumetric niacin response in male patients with schizophrenia who have seriously and dangerously violently offended: a (31)P magnetic resonance spectroscopy study

The aim of the study was to examine the association of arachidonic acid-related signal transduction with cerebral metabolism in patients with schizophrenia who have violently and dangerously offended while psychotic. Cerebral 31-phosphorus magnetic resonance spectroscopy was carried out in 11 male patients with schizophrenia who had violently offended (homicide, attempted murder, or wounding with intent to cause grievous bodily harm) while psychotic. Spectra were obtained from 70 x 70 x 70 mm(3) voxels using an image-selected in vivo spectroscopy pulse sequence. Niacin flush testing results were quantified as the volumetric niacin response. There was a strong, and negative, correlation between the volumetric niacin response and the metabolite concentration of inorganic phosphate expressed as a ratio of the total 31-phosphorus signal (p<0.005). Our results suggest that patients with schizophrenia who have violently offended and have poor phospholipid-related signal transduction may have higher levels of cerebral energy metabolism.     

31P-Nuclear magnetic resonance spectroscopy study of the time course of energy metabolism during exercise and recovery

This study evaluated the time courses of intracellular pH and the metabolism of phosphocreatine (PCr) and inorganic phosphate (P) at the onset of four exercise intensities and recoveries. Non-invasive evaluation of continuous changes in phosphorus metabolites has become possible using³¹P-nuclear magnetic resonance spectroscopy (³¹P-MRS). After measurements at rest, six healthy male subjects performed 4 min of femoral flexion exercise at intensities of 0 (loadless), 10, 20 and 30 kg · m · min^–1 in a 2.1 T superconducting magnet with a 67-cm bore. Measurements were continuously made during 5 min of recovery. During a series of rest-exercise-recovery procedures,³¹P-MRS were accumulated using 32 scans · spectrum^–1 requiring 12.8 s each. At the onset of exercise, PCr decreased exponentially with a time constant of 27–32 s regardless of the exercise intensity. The time constant PCr resynthesis during recovery was about 27–40 s. The PCr kinetics were independent of exercise intensity. There were similar Pi kinetics at the onset of all types of exercise, while those of Pi recovery became significantly longer at the higher exercise intensities (P < 0.05). Furthermore, the intracellular pH indicated temporary alkalosis just at the onset of exercise, probably due to absorption of hydrogen ions by PCr hydrolysis, and then decrease at a point about 40%–50% of the preexercise PCr. The pH recovery time was longer than that for the P_i or PCr kinetics. By using a more efficient resolution system it was possible to obtain the phosphorus kinetics during exercise and to follow PCr resynthesis within the first few minutes of recovery. From our results it was concluded that in general the time course of PCr and Pi metabolism were unaffected by the exercise intensity, both at the onset of exercise and during recovery, with the exception of P_i recovery.     

Exercise-induced splitting of the inorganic phosphate peak: investigation by time-resolved 31P-nuclear magnetic resonance spectroscopy

To investigate the splitting of the inorganic phosphate (Pi) peak during exercise and recovery, a time-resolved ³¹phosphorus nuclear magnetic resonance spectroscopy (³¹P-MRS) technique was used. Seven healthy young sedentary male subjects performed knee flexion exercise in the prone position inside a 2.1-T magnet, with the surface coil for ³¹P-MRS being placed on the biceps femoris muscle. After a 1-min warm-up without loading, the exercise intensity was increased by 0.41 W at 15-s intervals until exhaustion, followed by a 5-min recovery period. The ³¹P-MRS were recorded every 5 s during the rest-exercise-recovery sequence. Computer-aided contour analysis and pixel imaging of the Pi and phosphocreatine peaks were performed. Five of the seven subjects showed two distinct Pi peaks during exercise, suggesting two different pH distributions in exercising muscle (high pH and low pH region). In these five subjects, the high-pH increased rapidly just after the onset of exercise, while the low-pH peak increased gradually approximately 60 s after the onset of exercise. During recovery, the disappearance of the high-pH peak was more rapid than that of the low-pH peak. These findings suggest that our method ³¹P-MRS provides a simple approach for studying the kinetics of the Pi peak and intramuscular pH during exercise and recovery.     

A phosphorus-31 nuclear magnetic resonance study on the complex of chicken gizzard myosin subfragment 1 with adenosine diphosphate

The complex of Mg-ADP with chicken gizzard myosin subfragment 1 (S1), obtained by the treatment with Staphylococcus aureus V8 proteinase, was observed with 31P NMR at various temperatures between 0 and 25°C. The signal of S1·ADP complex was observed at -2 to -3 ppm as a rather broad peak. As compared with the results for rabbit skeletal muscle S1·ADP complex (Tanokura M, Ebashi S: J Biochem 113: 19-21, 1993), the signal was assigned to -phosphate of ADP in the S1·ADP complex. The signal of the complex was so broad and weak that the dependences on temperature and magnetic field strength were not clear. The observation suggests the tight interaction of S1 with the phosphate moieties of ADP in the complex and the extremely anisotropic distribution of electrons around phosphorus nuclei.     

31P-NMR study of pig intestinal brush-border membrane structure: effect of zinc and cadmium ions

³¹P-NMR experiments on intact pig small intestine brush-border membrane vesicles (BBMV) and detergent-solubilized membranes gave direct insights into the organization of the phospholipids (PL) and their interaction with zinc and cadmium ions. Various endogenous PL were identified from well resolved BBM micelle spectra. These experiments revealed a strong interaction of Zn²⁺ and Cd²⁺ with the negatively charged phosphatidylinositol and phosphatidylserine. In BBM micelles, a progressive time-dependent PL degradation occurred in the absence of ions and indicated the presence of active phospholipases. The presence of zinc inhibited the degradation process whereas cadmium had the opposite influence. ³¹P spectra of BBMV were carefully characterized. Neither zinc nor cadmium affected the PL bilayer structural organization. A degradation of PL, monitored by the increase of the inorganic phosphate (P _i) signal, also occurred in vesicles but to a lesser extent than in micelles. A 2/3 internal, 1/3 external PL asymmetry was observed in the absence and presence of ions. Offprint requests to: P. Ripoche     

Non-invasive assessment of oxidative capacity in young Indian men and women: a 31P magnetic resonance spectroscopy study

It is generally assumed that men display greater strength and muscle capacity than women. However, previous biochemical and histological studies have shown that men have greater capacity for anaerobic metabolism and women have higher or similar oxidative metabolism. Therefore, in the present study, we estimated oxidative capacity of gastrocnemius muscle and compared in Indian men and women using non-invasive in vivo 31P magnetic resonance spectroscopy (MRS). Healthy subjects (8 young males and 9 females, age-matched) performed plantar flexion exercise within a magnet and MRS measurements of inorganic phosphate (Pi), phosphocreatine (PCr), ADP, and pH of the calf muscles were carried out using a 1.5 T whole-body MRI system. PCr values during recovery were fitted to an exponential curve, and oxidative capacity was calculated using rate constant (k(PCr)), as an index of oxidative phosphorylation. When men and women were compared for different metabolic ratios, ADP, pH, k(PCr) and oxidative capacity, all parameters turned out to be statistically insignificant. The results showed no gender effect on skeletal muscle oxidative metabolism. The study demonstrated the usefulness of such non-invasive method to indirectly measure the oxidative capacity of the muscle based on PCr recovery.     

Phosphorus-31 nuclear magnetic resonance study of polyphosphate metabolism in intact ectomycorrhizal fungi

Summary Phosphorus-31 nuclear magnetic resonance spectra at 36.4 MHz are presented for intact ectomycorrhizal fungi grown in pure culture. Resonances from polyphosphates and intracellular orthophosphate are identified inCenococcum graniforme, Hebeloma cylindrosporum, andH. crustuliniforme. Comparison of the NMR spectra with phosphorus fractionation of the fungi extracts leads to the statement that the NMR-observed polyphosphaes is a good part of the accumulated polyphosphates. In actively growing mycelia, this fraction account for up to 17% of total P.     

Analysis of CHO-K1 cell growth in a fixed bed bioreactor using magnetic resonance spectroscopy and imaging

Non-invasive magnetic resonance imaging and spectroscopy techniques have been used to monitor the growth and distribution of Chinese hamster ovary K1 cells growing in a fixed bed bioreactor composed of macroporous carriers. Diffusion-weighted 1H magnetic resonance spectroscopy was used to monitor the volume fraction of the bioreactor occupied by the cells and diffusion-weighted 1H magnetic resonance imaging was used to map cell distribution. The imaging measurements demonstrated that cell growth in the bioreactor was heterogeneous, with the highest cell densities being found at the surface of the carriers. The increase in the volume fraction occupied by the cells during cell growth showed a close correlation with bioreactor ATP content measured using 31P magnetic resonance spectroscopy. These magnetic resonance measurements, in conjunction with measurements of bioreactor glucose consumption, allowed estimation of the specific glucose consumption rate. This declined during the culture, in parallel with medium glucose concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

The ultrastructure of the oyster brown cell,a cell with a fenestrated plasma membrane

Summary The oyster brown cell, a connective tissue cell of uncertain function and affinity, was characterized in the electron microscope by (1) the presence of large cytoplasmic granules, (2) fenestrations of the plasma membrane, and (3) an extensive tubular network originating in, or emptying into, the plasma membrane fenestrations. The brown cell did not appear to be a cell involved in glycogen storage or in the manufacture of exportable protein. The extensive tubular network and the membrane slits suggested that the brown cell may have been involved in the processing of biological fluids.This work was supported in part by Public Health Service Contract No. 5 To 1 ES00038-02, Health Sciences Advancement Award No. RR06138, and Tumor Biology Training Grant, NIH CA 05245.We wish to thank Miss Grete Nilsen for her expert technical assistance and Mr. Bob Munn for his help in the use of the electron microscope and for proof reading our MS. Our appreciation is also extended to Dr. J. Luft, Dr. A. K. Sparks, Miss P. Phelps, Mr. M. DeVault, and to the personnel of the Johnson Oyster Company, Inverness.     

Theoretical and experimental study of the membrane pore effect on the amplitude-frequency dependence of polarizability of a cell

The amplitude-frequency dependence of the polarizability of erythrocytes, yeast cells, and latex particles in the range of 1–10⁶ Hz was studied by the method of dielectrophoresis (DEP). Positive DEP of erythrocytes and yeast cells in a frequency range of 60–100 Hz was revealed. The positive DEP of cells in the given range is theoretically explained by appearance of a great number of transverse pores through the membrane and wall of the cell.     

Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance

 It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing. Both electron microscopy and broad-band ³¹P NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters ≤30 nm) and a population of larger vesicles with a frequency decreasing exponentially as the diameter increases. From ³¹P NMR investigations we inferred that the average diameter of DOPC/DOPA vesicles calculated on the basis of an exponential size distribution was of the order of 100 nm after 10 freeze-thaw cycles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawing does not have the same efficiency for all lipid mixtures. As found already by others, fragmentation into small vesicles requires the presence of salt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. If applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusion of sonicated unilamellar vesicles into larger vesicles only in pure water. These experiments show the usefulness of NMR as a complementary technique to electron microscopy for size determination of lipid vesicles. The applicability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles. Received: 2 September 1999 / Revised version: 27 February 2000 / Accepted: 27 February 2000     

Metabolic differentiation of Arabidopsis treated with methyl jasmonate using nuclear magnetic resonance spectroscopy

NMR spectroscopy combined with principal component analysis was applied to Arabidopsis thaliana treated with methyl jasmonate in order to obtain macroscopic metabolic changes caused by the treatment. As the first step several chromatographic and NMR spectroscopic techniques were utilized to identify metabolites of Arabidopsis. Sephadex LH-20 showed a high efficiency in the separation of phenolic metabolites in the plant. For identification of minor metabolites two-dimensional J-resolved NMR technique was directly applied to the plant extract and results in a number of elucidation of the metabolites of which signals overlap in ¹H NMR spectra. The chemical structure of the identified metabolites were confirmed by various two-dimensional NMR spectroscopy including correlated spectroscopy, heteronuclear single quantum coherence, and heternuclear multiple bond correlation. As next step, a statistical approach, principal component analysis based on projected J-resolved NMR spectra was performed for metabolic alteration of methyl jasmonate-treated Arabidopsis. The results show that methyl jasmonate caused an increase of flavonoids, fumaric acid, sinapoyl malate, sinigrin, tryptophan, valine, threonine, and alanine and a decrease of malic acid, feruloyl malate, glutamine, and carbohydrates after 24 h treatment.     

Temperature dependence of ultrasound-induced cell killing: The role of membrane fluidity

Chinese hamster cells in suspension were exposed to 20 kHz ultrasound (US) at 54 W/cm² and various temperatures between 2 and 44 °C. Activation energies were 2.6 and 24 kcal/mole below and above 35 °C, respectively. Procaine, a local anaesthetic drug known to increase membrane fluidity, enhanced cellular inactivation by US above 41 °C, increasing the activation energy to 62 kcal/mole. The inactivation of the bacterium Salmonella typhimurium by US was also dependent on the exposure temperature, with an activation energy of 2.9 kcal/mole between 2 and 44 °C. These data are most simply explained by the hypothesis that membranes are a major target for cellular inactivation by US and that the fluidity of the membranes is important in this respect.     

Phosphorus-31 nuclear magnetic resonance study on the effects of endurance training in rat skeletal muscle

To evaluate changes in muscle energetics following endurance training, we measured phosphorus-31 nuclear magnetic resonance (31P NMR) spectra on rat muscle in vivo before and after training in the same animals. The endurance training lasted for 3 months. The 31P NMR spectra were obtained serially at rest, during exercise by electrical stimulation, and during recovery. Intramuscular phosphocreatine (PCr), inorganic phosphate (P(i)), adenosine 5'-triphosphate (ATP) and pH were determined from the NMR spectra. The ratio of PCr:(PCR + P(i) at rest showed no difference between the trained and control groups even after 3 months of training. During exercise, however, this ratio was significantly higher in the trained group than in the control group. The ratio also recovered more rapidly after exercise in the trained group. The intramuscular pH decreased slightly by approximately 0.1 pH unit during exercise but did not show a significant difference between the groups. These results indicated that endurance training of 3 months duration improved the ATP supply system in the muscle. They also demonstrated that 31P NMR is a potent method for evaluating the effects of training in the same individuals.