pH-Dependent Piecewise Linear Correlation of 1H,31P Chemical Shifts: Application in NMR Identification of Nerve Agent Metabolites in Urine Samples.

The NMR-observable nuclei of the acidic and basic compounds experience pH dependence in chemical shift. This phenomenon can be exploited in NMR titrations to determine p Ka values of compounds, or in pH measurement of solutions using dedicated pH reference compounds. On the other hand, this sensitivity can also cause problems in, for example, metabolomics, where slight changes in pH result in significant difficulties for peak alignment between spectra of set of samples for comparative analysis. In worst case, the pH sensitivity of chemical shifts can prevent unambiguous identification of compounds. Here, we propose an alternative approach for NMR identification of pH-sensitive analytes. The 1H and X (13C, 15N, 31P, ...) chemical shifts in close proximity to the acidic or basic functional group should, when presented as ordered pairs, express piecewise linear correlation with distinct slope, intercept, and range. We have studied the pH dependence of 1H and 31P chemical shifts of the CH3-P moiety in urinary metabolites of nerve agents sarin, soman and VX using 2D 1H-31P fast-HMQC spectroscopy. The 1H and 31P chemical shifts of these chemicals appear in very narrow range, and due to subtle changes in sample pH the identification on either 1H or 31P chemical shift alone is uncertain. However, if the observed 1H and 31P chemical shifts of the CH3-P moiety of individual compounds are presented as ordered pairs, they fall into distinct linear spaces, thus, facilitating identification with high confidence.

Efficient 31 P band inversion transfer approach for measuring creatine kinase activity, ATP synthesis, and molecular dynamics in the human brain at 7 T.

PURPOSE: To develop an efficient 31 P magnetic resonance spectroscopy (MRS) method for measuring creatine kinase (CK) activity, adenosine triphosphate (ATP) synthesis, and motion dynamics in the human brain at 7 Tesla (T). METHODS: Three band inversion modules differing in center frequency were used to induce magnetization transfer (MT) effect in three exchange pathways: (i) CK-mediated reaction PCr → γ-ATP; (ii) de novo ATP synthesis Pi → γ-ATP; and (iii) ATP intramolecular 31 P-31 P cross-relaxation γ-(α-) ↔ ß-ATP. The resultant MT data were analyzed using a 5-pool model in the format of magnetization matrix according to Bloch-McConnell-Solomon formalism. RESULTS: With a repetition time (TR) of 4 s, the scan time for each module was approximately 8 min. The rate constants were kPCr → γATP 0.38 ± 0.02 s-1 , kPi → γATP 0.19 ± 0.02 s-1 , and σγ(α) ↔ ßATP 0.19 ± 0.04 s-1 , corresponding to ATP rotation correlation time τc (0.8 ± 0.2) ·10-7 s. The T1 relaxation times were Pi 7.26 ± 1.76 s, PCr 5.99 ± 0.58 s, γ-ATP 0.98 ± 0.07 s, α-ATP 0.95 ± 0.04 s, and ß-ATP 0.68 ± 0.03 s. CONCLUSION: Short-TR band inversion modules provide a time-efficient way of measuring brain ATP metabolism and could be useful in studying metabolic disorders in brain diseases. Magn Reson Med 78:1657-1666, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

In-vivo31P-MRS of skeletal muscle and liver: A way for non-invasive assessment of their metabolism.

In addition to direct assessment of high energy phosphorus containing metabolite content within tissues, phosphorus magnetic resonance spectroscopy (31P-MRS) provides options to measure phospholipid metabolites and cellular pH, as well as the kinetics of chemical reactions of energy metabolism in vivo. Even though the great potential of 31P-MR was recognized over 30 years ago, modern MR systems, as well as new, dedicated hardware and measurement techniques provide further opportunities for research of human biochemistry. This paper presents a methodological overview of the 31P-MR techniques that can be used for basic, physiological, or clinical research of human skeletal muscle and liver in vivo. Practical issues of 31P-MRS experiments and examples of potential applications are also provided. As signal localization is essential for liver 31P-MRS and is important for dynamic muscle examinations as well, typical localization strategies for 31P-MR are also described.

Dynamic PCr and pH imaging of human calf muscles during exercise and recovery using (31) P gradient-Echo MRI at 7 Tesla.

PURPOSE: Simultaneous acquisition of spatially resolved (31) P-MRI data for evaluation of muscle specific energy metabolism, i.e., PCr and pH kinetics. METHODS: A three-dimensional (3D) gradient-echo sequence for multiple frequency-selective excitations of the PCr and Pi signals in an interleaved sampling scheme was developed and tested at 7 Tesla (T). The pH values were derived from the chemical shift-induced phase difference between the resonances. The achieved spatial resolution was ∼2 mL with image acquisition time below 6 s. Ten healthy volunteers were studied performing plantar flexions during the delay between (31) P-MRI acquisitions, yielding a temporal resolution of 9-10 s. RESULTS: Signal from anatomically matched regions of interest had sufficient signal-to-noise ratio to allow single-acquisition PCr and pH quantification. The Pi signal was clearly detected in voxels of actively exercising muscles. The PCr depletions were in gastrocnemius 42 ± 14% (medialis), 48 ± 17% (lateralis) and in soleus 20 ± 11%. The end exercise pH values were 6.74 ± 0.18 and 6.65 ± 0.27 for gastrocnemius medialis and lateralis, respectively, and 6.96 ± 0.12 for soleus muscle. CONCLUSION: Simultaneous acquisition of PCr and Pi images with high temporal resolution, suitable for measuring PCr and pH kinetics in exercise-recovery experiments, was demonstrated at 7T. This study presents a fast alternative to MRS for quantifying energy metabolism of posterior muscle groups of the lower leg. Magn Reson Med 75:2324-2331, 2016. © 2015 Wiley Periodicals, Inc.

Involvement of Sac1 phosphoinositide phosphatase in the metabolism of phosphatidylserine in the yeast Saccharomyces cerevisiae.

Sac1 is a phosphoinositide phosphatase that preferentially dephosphorylates phosphatidylinositol 4-phosphate. Mutation of SAC1 causes not only the accumulation of phosphoinositides but also reduction of the phosphatidylserine (PS) level in the yeast Saccharomyces cerevisiae. In this study, we characterized the mechanism underlying the PS reduction in SAC1-deleted cells. Incorporation of (32) P into PS was significantly delayed in sac1∆ cells. Such a delay was also observed in SAC1- and PS decarboxylase gene-deleted cells, suggesting that the reduction in the PS level is caused by a reduction in the rate of biosynthesis of PS. A reduction in the PS level was also observed with repression of STT4 encoding phosphatidylinositol 4-kinase or deletion of VPS34 encoding phophatidylinositol 3-kinase. However, the combination of mutations of SAC1 and STT4 or VPS34 did not restore the reduced PS level, suggesting that both the synthesis and degradation of phosphoinositides are important for maintenance of the PS level. Finally, we observed an abnormal PS distribution in sac1∆ cells when a specific probe for PS was expressed. Collectively, these results suggested that Sac1 is involved in the maintenance of a normal rate of biosynthesis and distribution of PS.

Anodal transcranial direct current stimulation increases brain intracellular pH and modulates bioenergetics.

Transcranial direct current stimulation is an emerging treatment for brain disorders but its mode of action is not well understood. We applied 10 min 1 mA anodal transcranial direct current stimulation (tDCS) inside the bore of a 3 T MRI scanner to the left dorsolateral prefrontal cortex of 13 healthy volunteers (aged 19-28 yr) in a blinded, sham-controlled, cross-over design. Brain bioenergetics were measured from the left temporo-frontal region using 31P magnetic resonance spectroscopy before, during and for 20 min following tDCS. Brain pH rose during tDCS and remained elevated afterwards. Phosphomonoesters were significantly decreased while inorganic phosphate (Pi) also fell. Partial-least squares discriminant analysis of the data revealed two significantly different subject groups: one where phosphocreatine (PCr), ATP and Pi fell along with a larger increase in pH and one where PCr and ATP increased along with a smaller increase in pH and a slower and more sustained decrease in Pi. Group membership was predicted by baseline pH and ATP. We interpreted the effects of tDCS as driving two biochemical processes: cellular consumption of ATP causing hydrolysis of PCr via the creatine kinase reaction driving the increase in pH; synthesis of ATP and PCr by mitochondria with concomitant drop in Pi and phosphomonoester levels.

Abnormal response to cortical activation in early stages of Huntington disease.

BACKGROUND: We wished to identify noninvasive in vivo biomarkers of brain energy deficit in Huntington disease. METHODS: We studied 15 early affected patients (mean motor United Huntington Disease Rating Scale, 18 ± 9) and 15 age- and sex-matched controls. We coupled (31)phosphorus nuclear magnetic resonance spectroscopy with activation of the occipital cortex in order to measure the relative concentrations of adenosine triphosphate, phosphocreatine, and inorganic phosphate before, during, and after visual stimulation. RESULTS: In controls, we observed an 11% increase in the inorganic phosphate/phosphocreatine ratio (P = .024) and a 13% increase in the inorganic phosphate/adenosine triphosphate ratio (P = .016) during brain activation, reflecting increased adenosine diphosphate concentrations. Subsequently, controls had a return to baseline levels during recovery (P = .012 and .022, respectively). In contrast, both ratios were unchanged in patients during and after visual stimulation. CONCLUSIONS: (31)Phosphorus nuclear magnetic resonance spectroscopy could provide functional biomarkers of brain energy deficit to monitor therapeutic efficacy in Huntington disease.

Altered relative concentrations of high-energy phosphates in patients with uraemic cardiomyopathy measured by magnetic resonance spectroscopy.

BACKGROUND: Premature sudden cardiovascular death is the commonest cause of death in end-stage renal disease (ESRD) patients and is associated with uraemic cardiomyopathy [left ventricular hypertrophy (LVH), systolic dysfunction (LVSD) or LV dilation]. High-energy phosphates (HEP), quantified using phosphorus-31 magnetic resonance spectroscopy, are reduced in patients with diabetes, heart failure and uraemia. Phosphocreatine:ß adenosine triphosphate (PCr:ATP) ratio is an index of metabolic activity. We compared resting HEPs in ESRD patients and hypertensive patients (with and without LVH) who had normal renal function (LVH-only or normal myocardia). We also assessed associations of HEP levels with abnormalities of uraemic cardiomyopathy. METHODS: Fifty-three ESRD and 30 hypertensive patients (18 with LVH, 12 with normal myocardia) underwent phosphorus magnetic resonance spectroscopy of their left ventricle. PCr:ATP ratios were calculated from (31)P-MR spectra obtained from long-axis views of the left ventricle. RESULTS: There were no significant differences in age, LV mass, chamber sizes and ejection fraction between patient groups. PCr:ATP was significantly lower in ESRD patients compared to hypertensive patients, irrespective of the presence or absence of LVH (P = 0.01). In the ESRD group, PCr:ATP was significantly lower in patients with LVSD (P = 0.05) and LV dilation (P = 0.01). LVH was not associated with significant difference in PCr:ATP. CONCLUSIONS: ESRD patients have lower HEP levels compared to hypertensive patients. Lower PCr:ATP ratio, indicating altered myocardial metabolic function in ESRD patients, is associated with features of uraemic cardiomyopathy.

Use of 1H and 31P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies.

Quantitative multinuclear high-resolution magic angle spinning was performed in order to determine the tissue pH values of and the absolute metabolite concentrations in 33 samples of human brain tumour tissue. Metabolite concentrations were quantified by 1D (1)H and (31)P HRMAS using the electronic reference to in vivo concentrations (ERETIC) synthetic signal. (1)H-(1)H homonuclear and (1)H-(31)P heteronuclear correlation experiments enabled the direct assessment of the (1)H-(31)P spin systems for signals that suffered from overlapping in the 1D (1)H spectra, and linked the information present in the 1D (1)H and (31)P spectra. Afterwards, the main histological features were determined, and high heterogeneity in the tumour content, necrotic content and nonaffected tissue content was observed. The metabolite profiles obtained by HRMAS showed characteristics typical of tumour tissues: rather low levels of energetic molecules and increased concentrations of protective metabolites. Nevertheless, these characteristics were more strongly correlated with the total amount of living tissue than with the tumour cell contents of the samples alone, which could indicate that the sampling conditions make a significant contribution aside from the effect of tumour development in vivo. The use of methylene diphosphonic acid as a chemical shift and concentration reference for the (31)P HRMAS spectra of tissues presented important drawbacks due to its interaction with the tissue. Moreover, the pH data obtained from (31)P HRMAS enabled us to establish a correlation between the pH and the distance between the N(CH(3))(3) signals of phosphocholine and choline in (1)H spectra of the tissue in these tumour samples.

Electron spray ionization mass spectrometry and 2D 31P NMR for monitoring 18O/16O isotope exchange and turnover rates of metabolic oligophosphates.

A new method was here developed for the determination of (18)O-labeling ratios in metabolic oligophosphates, such as ATP, at different phosphoryl moieties (α-, ß-, and γ-ATP) using sensitive and rapid electrospray ionization mass spectrometry (ESI-MS). The ESI-MS-based method for monitoring of (18)O/(16)O exchange was validated with gas chromatography-mass spectrometry and 2D (31)P NMR correlation spectroscopy, the current standard methods in labeling studies. Significant correlation was found between isotopomer selective 2D (31)P NMR spectroscopy and isotopomer less selective ESI-MS method. Results demonstrate that ESI-MS provides a robust analytical platform for simultaneous determination of levels, (18)O-labeling kinetics and turnover rates of α-, ß-, and γ-phosphoryls in ATP molecule. Such method is advantageous for large scale dynamic phosphometabolomic profiling of metabolic networks and acquiring information on the status of probed cellular energetic system.

A new experimental approach and signal processing scheme for the detection and quantitation of ³¹P brain neurochemicals from in vivo MRS studies using dual tuned (¹H/³¹P) head coil.

Brain (31)P-neurometabolites play an important role in energy and membrane metabolism. Unambiguous identification and quantification of these neurochemicals in different brain regions would be a great aid in advancing the understanding of metabolic processes in the nervous system. Phosphomonoester (PME), consisting of phosphoethanolamine (PE) and phosphocholine (PC), is the "building block" for membranes, while phosphodiesters (PDE), consisting of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) metabolites are involved in the membrane breakdown process. In the clinical setting, generating well-resolved spectra for PC, PE, GPC, and GPE could be crucial phospholipids in providing information regarding membrane metabolism. We present here a new experimental approach for generating well-resolved (31)P spectra for PC and PE as well as for GPC, GPE, and other (31)P metabolites. Our results (based on uni-dimensional (1D) and multi-voxel (31)P studies) indicate that an intermediate excitation pulse angle (35°) is best suited to obtain well-resolved PC/PE and GPC/GPE resonance peaks. Our novel signal processing scheme allows generating metabolite maps of different phospholipids include PC/PE and GPC/GPE using the 'time-domain-frequency-domain' method as referred to in the MATLAB programming language.

A comparison of 31P magnetic resonance spectroscopy and microbubble-enhanced ultrasound for characterizing hepatitis c-related liver disease.

We compared in vivo hepatic (31) P magnetic resonance spectroscopy ((31) P MRS) and hepatic vein transit times (HVTT) using contrast-enhanced ultrasound with a microbubble agent to assess the severity of hepatitis C virus (HCV)-related liver disease. Forty-six patients with biopsy-proven HCV-related liver disease and nine healthy volunteers had (31) P MRS and HVTT performed on the same day. (31) P MR spectra were obtained at 1.5 T. Peak areas were calculated for metabolites, including phosphomonoesters (PME) and phosphodiesters (PDE). Patients also had the microbubble ultrasound contrast agent, Levovist (2 g), injected into an antecubital vein, and time-intensity Doppler ultrasound signals of the right and middle hepatic veins were measured. The HVTT was calculated as the time from injection to a sustained rise in Doppler signal 10% greater than baseline. The shortest times were used for analysis. Based on Ishak histological scoring, there were 15 patients with mild hepatitis, 20 with moderate/severe hepatitis and 11 with cirrhosis. With increasing severity of disease, the PME/PDE ratio was steadily elevated, while the HVTT showed a monotonic decrease. Both imaging modalities could separate patients with cirrhosis from the mild and moderate/severe hepatitis groups. No statistical difference was observed in the accuracy of each test to denote mild, moderate/severe hepatitis and cirrhosis (Fisher's exact test P =1.00). (31) P MRS and HVTT show much promise as noninvasive imaging tests for assessing the severity of chronic liver disease. Both are equally effective and highly sensitive in detecting cirrhosis.

Measurement of creatine kinase reaction rate in human brain using magnetization transfer image-selected in vivo spectroscopy (MT-ISIS) and a volume ³¹P/¹H radiofrequency coil in a clinical 3-T MRI system.

High-energy phosphate metabolism, which allows the synthesis and regeneration of adenosine triphosphate (ATP), is a vital process for neuronal survival and activity. In particular, creatine kinase (CK) serves as an energy reservoir for the rapid buffering of ATP levels. Altered CK enzyme activity, reflecting compromised high-energy phosphate metabolism or mitochondrial dysfunction in the brain, can be assessed using magnetization transfer (MT) MRS. MT (31)P MRS has been used to measure the forward CK reaction rate in animal and human brain, employing a surface radiofrequency coil. However, long acquisition times and excessive radiofrequency irradiation prevent these methods from being used routinely for clinical evaluations. In this article, a new MT (31)P MRS method is presented, which can be practically used to measure the CK forward reaction rate constant in a clinical MRI system employing a volume head (31)P coil for spatial localization, without contamination from the scalp muscle, and an acquisition time of 30 min. Other advantages associated with the method include radiofrequency homogeneity within the regions of interest of the brain using a volume coil with image-selected in vivo spectroscopy localization, and reduction of the specific absorption rate using nonadiabatic radiofrequency pulses for MT saturation. The mean value of k(f) was measured as 0.320 ± 0.075 s(-1) from 10 healthy volunteers with an age range of 18-40 years. These values are consistent with those obtained using earlier methods, and the technique may be used routinely to evaluate energetic processes in the brain on a clinical MRI system.

A comparison of single-voxel clinical in vivo hepatic 31P MR spectra acquired at 1.5 and 3.0 Tesla in health and diseased states.

With the increasing availability of human MR scanners at various field strengths, the optimal field strength for in vivo clinical MR studies of the liver has become a focus of attention. Comparison between results at 3.0 and 1.5 T is of particular clinical interest, especially for multicentre studies. For MRS studies, higher field strengths should be advantageous, because improved sensitivity and chemical shift dispersion are expected. We report a comparison between single-voxel hepatic proton-decoupled (31)P MRS performed at 1.5 and 3.0 T in the same subjects using similar methodologies. Twelve healthy volunteers and 15 patients with chronic liver disease were studied. Improved spectral resolution was achieved using proton decoupling, and there was an improvement (21%) in the signal-to-noise ratio (SNR) of the phosphomonoester (PME) resonance at 3.0 T relative to 1.5 T. There was no significant change in nuclear Overhauser effects for PME or phosphodiesters (PDEs) between the two field strengths. The T(1) value of PDE was significantly longer at 3 T, although there was no significant change in the T(1) value of PME. There was no significant difference in the mean PME/PDE ratios for either the control or patient groups at both 1.5 and 3.0 T, but there was a small positive mean difference in PME/PDE at 3.0 T on pairwise testing between field strengths (+ 0.05, p < 0.01). There were significant correlations between PME/PDE values at the two magnetic field strengths (r = 0.806, p < 0.001). The underlying broad resonance was reduced at 3.0 T relative to 1.5 T, related to line broadening of the phospholipid bilayer signal. In conclusion, there was an improvement in hepatic (31)P MR signal quality at 3.0 T relative to 1.5 T. Broadly similar hepatic (31)P MR parameters were obtained at 1.5 and 3.0 T. The modest difference noted in the PME/PDE ratio between field strengths for patients with chronic liver disease should inform multicentre study design involving these field strengths.

In vivo 31P MRS detection of an alkaline inorganic phosphate pool with short T1 in human resting skeletal muscle.

Non-invasive determination of mitochondrial content is an important objective in clinical and sports medicine. 31P MRS approaches to obtain information on this parameter at low field strength typically require in-magnet exercise. Direct observation of the intra-mitochondrial inorganic phosphate (Pi) pool in resting muscle would constitute an alternative, simpler method. In this study, we exploited the higher spectral resolution and signal-to-noise at 7T to investigate the MR visibility of this metabolite pool. 31P in vivo MR spectra of the resting soleus (SOL) muscle were obtained with 1H MR image-guided surface coil localization (six volunteers) and of the SOL and tibialis anterior (TA) muscle using 2D CSI (five volunteers). A resonance at a frequency 0.38 ppm downfield from the cytosolic Pi resonance (Pi(1); pH 7.0 ± 0.04) was reproducibly detected in the SOL muscle in all subjects and conditionally attributed to the intra-mitochondrial Pi pool (Pi(2); pH 7.3 ± 0.07). In the SOL muscle, the Pi(2)/Pi(1) ratio was 1.6 times higher compared to the TA muscle in the same individual. Localized 3D CSI results showed that the Pi(2) peak was present in voxels well away from blood vessels. Determination of the T1 of the two Pi pools in a single individual using adiabatic excitation of the spectral region around 5 ppm yielded estimates of 4.3 ± 0.4 s vs 1.4 ± 0.5 s for Pi(1) and Pi(2), respectively. Together, these results suggest that the intra-mitochondrial Pi pool in resting human skeletal muscle may be visible with 31P MRS at high field.

Measuring the acute effect of insulin infusion on ATP turnover rate in human skeletal muscle using phosphorus-31 magnetic resonance saturation transfer spectroscopy.

Mitochondrial dysfunction has been proposed to underlie the insulin resistance of type 2 diabetes. However, the relative time course of insulin action in stimulating ATP turnover rate and glucose uptake in skeletal muscle has not been examined. These two parameters were measured in young healthy subjects using the (31)P MRS saturation transfer method in conjunction with the euglycaemic hyperinsulinaemic clamp technique respectively. Glucose infusion rate rose rapidly from 0 to 2.90 ± 0.11 mg/kg(ffm)/min during the first 10 min of insulin infusion and further to 6.17 ± 0.57 mg/kg(ffm)/min between 15 and 45 min. In contrast, baseline ATP turnover rate was 9.0 ± 0.4 µmol/g/min of muscle and did not change during the first 45 min of insulin infusion. Between 50 and 80 minutes ATP turnover rate increased by 8% and remained steady to 150 minutes (9.7 ± 0.5 µmol/g/min of muscle, p = 0.03 vs baseline). The in vivo time course of insulin stimulation of skeletal muscle ATP turnover rate is not consistent with a rate limiting effect upon the initiation of insulin-stimulated glycogen synthesis.

31P magnetic resonance spectroscopic imaging with polarisation transfer of phosphomono- and diesters at 3 T in the human brain: relation with age and spatial differences.

Tissue levels of the compounds phosphocholine (PC), phosphoethanolamine (PE), glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) can be studied by in vivo 31P MRS. However, the detection of the signals of these compounds suffers from low sensitivity and contamination by underlying broad resonances of other phosphorylated compounds. Improved sensitivity without this contamination can be achieved with a method for optimal polarisation transfer of 1H to 31P spins in these molecules, called selective refocused insensitive nuclei-enhanced polarisation transfer (sRINEPT). The aim of this study was to implement a three-dimensional magnetic resonance spectroscopic imaging (MRSI) version of sRINEPT on a clinical 3 T magnetic resonance system to obtain spatially resolved relative levels of PC, PE, GPC and GPE in the human brain as a function of age, which could be used as a reference dataset for clinical applications. Good signal-to-noise ratios were obtained from voxels of 17 cm(3) of the parietal and occipital lobes of the brain within a clinically acceptable measurement time of 17 min. Eighteen healthy subjects of different ages (16-70 years) were examined with this method. A strong inverse relation of the PE/GPE and PC/GPC ratios with age was found. Spatial resolution was sufficient to detect differences in metabolite ratios between white and grey matter. Moreover, we showed the feasibility of this method for clinical use in a pilot study of patients with brain tumours. The sRINEPT MRSI technique enables the exploration of phospholipid metabolism in brain diseases with a better sensitivity than was possible with earlier 31P MRS methods.

Δ9-Tetrahydrocannabivarin suppresses in vitro epileptiform and in vivo seizure activity in adult rats.

PURPOSE: We assessed the anticonvulsant potential of the phytocannabinoid Δ9-tetrahydrocannabivarin (Δ9-THCV) by investigating its effects in an in vitro piriform cortex (PC) brain slice model of epileptiform activity, on cannabinoid CB1 receptor radioligand-binding assays and in a generalized seizure model in rats. METHODS: Δ9-THCV was applied before (10 µm Δ9-THCV) or during (10-50 µm Δ9-THCV) epileptiform activity induced by Mg²(+) -free extracellular media in adult rat PC slices and measured using multielectrode array (MEA) extracellular electrophysiologic techniques. The actions of Δ9-THCV on CB1 receptors were examined using [³H]SR141716A competition binding and [³5S]GTPγS assays in rat cortical membranes. Effects of Δ9-HCV (0.025-2.5 mg/kg) on pentylenetetrazole (PTZ)-induced seizures in adult rats were also assessed. RESULTS: After induction of stable spontaneous epileptiform activity, acute Δ9 -THCV application (≥ 20 µm) significantly reduced burst complex incidence and the amplitude and frequency of paroxysmal depolarizing shifts (PDSs). Furthermore, slices pretreated with 10 µm Δ9-THCV prior to induction of epileptiform activity exhibited significantly reduced burst complex incidence and PDS peak amplitude. In radioligand-binding experiments, Δ9-THCV acted as a CB1 receptor ligand, displacing 0.5 nm [³H]SR141716A with a Ki∼290 nm, but exerted no agonist stimulation of [³5S]GTPγS binding. In PTZ-induced seizures in vivo, 0.25 mg/kg Δ9-THCV significantly reduced seizure incidence. DISCUSSION: These data demonstrate that Δ9-THCV exerts antiepileptiform and anticonvulsant properties, actions that are consistent with a CB1 receptor-mediated mechanism and suggest possible therapeutic application in the treatment of pathophysiologic hyperexcitability states.

An intracellular pH gradient in the anammox bacterium Kuenenia stuttgartiensis as evaluated by 31P NMR.

The cytoplasm of anaerobic ammonium oxidizing (anammox) bacteria consists of three compartments separated by membranes. It has been suggested that a proton motive force may be generated over the membrane of the innermost compartment, the "anammoxosome". 31P nuclear magnetic resonance (NMR) spectroscopy was employed to investigate intracellular pH differences in the anammox bacterium Kuenenia stuttgartiensis. With in vivo NMR, spectra were recorded of active, highly concentrated suspensions of K. stuttgartiensis in a wide-bore NMR tube. At different external pH values, two stable and distinct phosphate peaks were apparent in the recorded spectra. These peaks were equivalent with pH values of 7.3 and 6.3 and suggested the presence of a proton motive force over an intracytoplasmic membrane in K. stuttgartiensis. This study provides for the second time--after discovery of acidocalcisome-like compartments in Agrobacterium tumefaciens--evidence for an intracytoplasmic pH gradient in a chemotrophic prokaryotic cell.

[Biochemical metabolic changes detected by phosphorus-31 MR spectroscopy in liver of fasting rabbits].

OBJECTIVE: To investigate the biochemical metabolic changes detected by phosphorus-31 MR spectroscopy ((31)P MRS) with pathologic changes in the liver of fasting rabbits. METHODS: A total of 22 rabbits were under the starvation up to death to establish animal models. Hepatic (31)P MRS was performed in different period of 10 rabbits including normal condition, over-starvation, agonal condition and death after 30 min. Other 9 rabbits were divided into three type including over-starvation, agonal condition and death group with 3 rabbits in each group, and 3 healthy rabbits served as controls. All the 12 rabbits were sacrificed for the hepatic pathological examination. The MR examination was performed on a 1.5 T imager using a 1H/31P surface coil by the 2D chemical shift imaging technique. The relative quantities of phosphomonoesters (PME), phosphodiesters (PDE), inorganic phosphate (Pi) and adenosine triphosphate (ATP) were measured. RESULTS: All the relative quantification of phosphorus metabolites were changed significantly from starvation to death (X(2)=23.13-35.41, P<0.01). The relative quantifications of ATP of normal condition, over-starvation, agonal condition and death were 2.54 +/-0.53, 1.73 +/-0.14, 0.88 +/-0.23 and 0.05 +/-0.08, respectively (rs=1.0, P<0.01). The relative quantifications of PDE from normal to death were 1.25 +/-0.54, 2.76 +/-0.23, 3.33 +/-0.49 and 3.87 +/-0.43, respectively, and those of Pi were 0.42 +/-0.02, 0.65 +/-0.05, 0.89 +/-0.15 and 0.99 +/-0.08, respectively (rs=1.0, P <0.01). The relative quantifications of PME were also significantly changed (rs=0.4, P=0.6). The pathologic changes of normal condition, over-starvation, agonal condition and death: decreased size of hepatocytes, loss of cell number, cellular swelling, degeneration and cell necrosis or hepatic hemorrhage became more and more pronounced. CONCLUSION: (31)P MRS can monitor dynamic changes of relative quantification of phosphorus metabolites, which are correlated with the pathological severity of acute hepatic injury by fasting.