Rapid In Vivo Quantification of Creatine Kinase Activity by Phosphorous-31 Magnetic Resonance Spectroscopic Fingerprinting (31P-MRSF).

Creatine kinase (CK) plays an important role in tissue metabolism by providing a buffering mechanism for maintaining a constant supply of adenosine triphosphate (ATP) during metabolic perturbations. Phosphorous-31 magnetic resonance spectroscopy (31P-MRS) employing magnetization transfer techniques is the only noninvasive method for measuring the rate of ATP synthesis via creatine kinase. However, due to the low concentrations of phosphate metabolites, current 31P-MRS methods require long acquisition time to achieve adequate measurement accuracy. In this chapter, we present a new framework of data acquisition and parameter estimation, the 31P magnetic resonance spectroscopic fingerprinting (31P-MRSF) method, for rapid quantification of CK reaction rate constant in the hindlimb of small laboratory animals.

Characterization of an archaeal inorganic pyrophosphatase from Sulfolobus islandicus using a [31P]-NMR-based assay.

Inorganic pyrophosphatase catalyzes the conversion of pyrophosphate to phosphate and is often critical for driving reactions forward in cellular processes such as nucleic acid and protein synthesis. Commonly used methods for quantifying pyrophosphatase enzyme activity employ reacting liberated phosphate with a second molecule to produce absorbance changes or employing a second enzyme in coupled reactions to produce a product with a detectable absorbance. In this investigation, a novel [31P]-NMR spectroscopy-based assay was used to quantitatively measure the formation of phosphate and evaluate the activity of inorganic pyrophosphatase from the thermoacidophilic Crenarchaeota Sulfolobus islandicus. The enzymatic activity was directly measured via integration of the [31P] resonance associated with the phosphate product (δ = 2.1 ppm). Sulfolobus islandicus inorganic pyrophosphatase preferentially utilized Mg2+ as divalent cation and had pH and temperature optimums of 6.0 of 50 °C, respectively. The Vmax value was 850 µmol/min/mg and the Km for pyrophosphate was 1.02 mM. Sequence analysis indicates the enzyme is a Family I pyrophosphatase. Sulfolobus islandicus inorganic pyrophosphatase was shown to be inhibited by sodium fluoride with a IC50 of 2.26 mM, compared to a IC50 of 0.066 mM for yeast inorganic pyrophosphatase. These studies reveal that a [31P]-NMR spectroscopy-based assay is an effective method for analyzing catalysis by phosphate-producing enzymes.

Repeated 31P-Magnetic Resonance Spectroscopy in Severe Traumatic Brain Injury: Insights into Cerebral Energy Status and Altered Metabolism.

Phosphorous magnetic resonance spectroscopy (31P-MRS) is suited to noninvasively investigate energy metabolism and to detect molecules containing phosphorus in the human brain. The aim of this longitudinal study was to perform 31P-MRS at two different time points (within 72 h and between day 10-14) after severe traumatic brain injury (sTBI) to reveal alterations in cerebral energy metabolism. Twenty-six ventilated patients with sTBI, aged between 20 and 75 years, with a median initial Glasgow Coma Scale score of 5 were analyzed prospectively. The 31P-MRS data of the structurally more affected side were compared with data from contralateral normal appearing areas and with data of age- and gender-matched healthy controls. There were no significant intraindividual differences between the lesioned and the less affected side at either of the time points. In the acute phase, phosphocreatine/adenosine triphosphate (PCr/ATP) and phosphocreatine/inorganic phosphate (PCr/Pi) were significantly elevated whereas phosphomonoesters/phosphodiesters (PME/PDE) and Pi/ATP were significantly decreased in contrast to healthy controls. In the subacute phase, these differences gradually dissipated, remaining lower Pi/ATP ratio, and only partly altered levels of PCr/Pi and PME/PDE. Our data affirm that cerebral metabolism is globally altered after sTBI, demonstrating the diffuse impairment of brain bioenergetics at multiple levels, with resultant developments in terms of time.

A pipeline for making 31P NMR accessible for small- and large-scale lipidomics studies.

Detailed molecular analysis is of increasing importance in research into the regulation of biochemical pathways, organismal growth and disease. Lipidomics in particular is increasingly sought after as it provides insight into molecular species involved in energy storage, signalling and fundamental cellular structures. This has led to the use of a range of tools and techniques to acquire lipidomics data. 31P NMR for lipidomics offers well-resolved head group/lipid class analysis, structural data that can be used to inform and strengthen interpretation of mass spectrometry data and part of a priori structural determination. In the present study, we codify the use of 31P NMR for lipidomics studies to make the technique more accessible to new users and more useful for a wider range of questions. The technique can be used in isolation (phospholipidomics) or as a part of determining lipid composition (lipidomics). We describe the process from sample extraction to data processing and analysis. This pipeline is important because it allows greater thoroughness in lipidomics studies and increases scope for answering scientific questions about lipid-containing systems.

Novel Mitochondria-Targeted Triphenylphosphonium Conjugates of Linear ß-Phosphorylated Nitrones : Preparation, 31P NMR Mitochondrial Distribution, EPR Spin Trapping Reporting, and Site-Directed Antiapoptotic Properties.

The mitochondrion can be considered as the metabolic powerhouse of the cell, having a key impact on energy production, cell respiration, and intrinsic cell death. Mitochondria are also the main source of endogenous reactive oxygen species , including free radicals (FR), which are physiologically involved in signaling pathways but may promote cell damage when unregulated or excessively formed in inappropriate locations. A variety of chronic pathologies have been associated with FR-induced mitochondrial dysfunctions , such as cancer, age-related neurodegenerative diseases, and metabolic syndrome.In recent years drug design based on specific mitochondria-targeted antioxidants has become a very attractive therapeutic strategy and, among target compounds, nitrones have received growing attention because of their specific affinity toward FR. Here, we describe protocols dealing with the preparation, mitochondria permeation assessment, electron paramagnetic resonance (EPR) spin trapping setting, and antiapoptotic properties evaluation of a series of new linear nitrones vectorized by a triphenylphosphonium cation and labeled with a diethoxyphosphoryl moiety as 31P nuclear magnetic resonance (NMR) reporter with antioxidant property.

Mefloquine synergism with anti-tuberculosis drugs and correlation to membrane effects: Biologic, spectroscopic and molecular dynamics simulations studies.

Studies displaying the combination of mefloquine (MFL) with anti-tuberculosis (TB) substances are limited in the literature. In this work, the effect of MFL-association with two first-line anti-TB drugs and six fluoroquinolones was evaluated against Mycobacterium tuberculosis drug resistant strains. MFL showed synergistic interaction with isoniazid, pyrazinamide, and several fluoroquinolones, reaching fractional inhibitory concentration indexes (FICIs) ranging from 0.03 to 0.5. In order to better understand the observed results, two approaches have been explored: (i) spectroscopic responses attributed to the effect of MFL on physicochemical properties related to a liposomal membrane model composed by soybean asolectin; (ii) molecular dynamics (MD) simulation data regarding MFL interaction with a membrane model based on PIM2, a lipid constituent of the mycobacterial cell wall. FTIR and NMR data showed that MFL affects expressively the region between the phosphate and the first methylene groups of soybean asolectin membranes, disordering these regions. MD simulations results detected high MFL density in the glycolipid interface and showed that the drug increases the membrane lateral diffusion, enhancing its permeability. The obtained results suggest that synergistic activities related to MFL are attributed to its effect of lipid disorder and membrane permeability enhancement.

Late post-natal neurometabolic development in healthy male rats using 1 H and 31 P magnetic resonance spectroscopy.

Brain metabolism evolves rapidly during early post-natal development in the rat. While changes in amino acids, energy metabolites, antioxidants or metabolites involved in phospholipid metabolism have been reported in the early stages, neurometabolic changes during the later post-natal period are less well characterized. Therefore, we aimed to assess the neurometabolic changes in male Wistar rats between post-natal days 29 and 77 (p29-p77) using longitudinal magnetic resonance spectroscopy (MRS) in vivo at 9.4 Tesla. 1 H MRS was performed in the hippocampus between p29 and p77 at 1-week intervals (n = 7) and in the cerebellum between p35 and p77 at 2-week intervals (n = 7) using the SPECIAL sequence at ultra-short echo-time. NOE enhanced and 1 H decoupled 31 P MR spectra were acquired at p35, p48 and p63 (n = 7) in a larger voxel covering cortex, hippocampus and part of the striatum. The hippocampus showed a decrease in taurine concentration and an increase in glutamate (with more pronounced changes until p49), seemingly a continuation of their well-described changes in the early post-natal period. A constant increase in myo-inositol and choline-containing compounds in the hippocampus (in particular glycero-phosphocholine as shown by 31 P MRS) was measured throughout the observation period, probably related to membrane metabolism and myelination. The cerebellum showed only a significant increase in myo-inositol between p35 and p77. In conclusion, this study showed important changes in brain metabolites in both the hippocampus and cerebellum in the later post-natal period (p29/p35-p77) of male rats, something previously unreported. Based on these novel data, changes in some neurometabolites beyond p28-35, conventionally accepted as the cut off for adulthood, should be taken into account in both experimental design and data interpretation in this animal model.

18O Isotope Labeling Combined with 31P Nuclear Magnetic Resonance Spectroscopy for Accurate Quantification of Hydrolyzable Phosphorus Species in Environmental Samples.

31P nuclear magnetic resonance (NMR) spectra can be biased due to the hydrolysis of labile P species during sample treatment and NMR analysis. This paper offers an approach to circumvent this problem by performing sample preparation and analysis in 18O-enriched medium. Heavy 18O isotope atoms were introduced into the resulting artificial hydrolysis products. The NMR signal of 18O-labeled P was shifted upfield relative to the unlabeled P nuclei in natural metabolites. This isotope shift enabled an immediate differentiation of artificial hydrolysis products from natural metabolites. Moreover, the hydrolysis products could be accurately quantified. Our data suggest that the extent to which artificial hydrolysis alters NMR spectra varies among different types of environmental samples. For instance, 72-84% of the detected monoesters in the organic soils of this study were actually artificially hydrolyzed diesters. By contrast, artificial hydrolysis products in the mineral soils used for this study accounted for less than 6% of the total monoesters. Polyphosphate was also hydrolyzed to yield 18O-labeled products in algal biomass.

Intracellular Phosphate and ATP Depletion Measured by Magnetic Resonance Spectroscopy in Patients Receiving Maintenance Hemodialysis.

BACKGROUND: The precise origin of phosphate that is removed during hemodialysis remains unclear; only a minority comes from the extracellular space. One possibility is that the remaining phosphate originates from the intracellular compartment, but there have been no available data from direct assessment of intracellular phosphate in patients undergoing hemodialysis. METHODS: We used phosphorus magnetic resonance spectroscopy to quantify intracellular inorganic phosphate (Pi), phosphocreatine (PCr), and ßATP. In our pilot, single-center, prospective study, 11 patients with ESKD underwent phosphorus (31P) magnetic resonance spectroscopy examination during a 4-hour hemodialysis treatment. Spectra were acquired every 152 seconds during the hemodialysis session. The primary outcome was a change in the PCr-Pi ratio during the session. RESULTS: During the first hour of hemodialysis, mean phosphatemia decreased significantly (-41%; P<0.001); thereafter, it decreased more slowly until the end of the session. We found a significant increase in the PCr-Pi ratio (+23%; P=0.001) during dialysis, indicating a reduction in intracellular Pi concentration. The PCr-ßATP ratio increased significantly (+31%; P=0.001) over a similar time period, indicating a reduction in ßATP. The change of the PCr-ßATP ratio was significantly correlated to the change of depurated Pi. CONCLUSIONS: Phosphorus magnetic resonance spectroscopy examination of patients with ESKD during hemodialysis treatment confirmed that depurated Pi originates from the intracellular compartment. This finding raises the possibility that excessive dialytic depuration of phosphate might adversely affect the intracellular availability of high-energy phosphates and ultimately, cellular metabolism. Further studies are needed to investigate the relationship between objective and subjective effects of hemodialysis and decreases of intracellular Pi and ßATP content. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Intracellular Phosphate Concentration Evolution During Hemodialysis by MR Spectroscopy (CIPHEMO), NCT03119818.

Sensitive Morphological Characterization of Oriented High-Density Lipoprotein Nanoparticles Using 31 P†NMR Spectroscopy.

The biological function of high-density lipoprotein (HDL) nanoparticles, the so-called good cholesterol that is associated with a low risk of heart disease, depends on their composition, morphology, and size. The morphology of HDL particles composed of apolipoproteins, lipids and cholesterol is routinely visualised by transmission electron microscopy (TEM), but higher-resolution tools are needed to observe more subtle structural differences between particles of different composition. Here, reconstituted HDL formulations are oriented on glass substrates and solid-state 31 P NMR spectroscopy is shown to be highly sensitive to the surface curvature of the lipid headgroups. The spectra report potentially functionally important differences in the morphology of different HDL preparations that are not detected by TEM. This method provides new morphological insights into HDL comprising a naturally occurring apolipoprotein A-I mutant, which may be linked to its atheroprotective properties, and holds promise as a future research tool in the clinical analysis of plasma HDL.

Creatine kinase rate constant in the human heart at 7T with 1D-ISIS/2D CSI localization.

PURPOSE: Creatine Kinase (CK) reaction plays an important role in energy metabolism and estimate of its reaction rate constant in heart provides important insight into cardiac energetics. Fast saturation transfer method ([Formula: see text] nominal) to measure CK reaction rate constant (kf) was previously demonstrated in open chest swine hearts. The goal of this work is to further develop this method for measuring the kf in human myocardium at 7T. [Formula: see text] approach is combined with 1D-ISIS/2D-CSI for in vivo spatial localization and myocardial CK forward rate constant was then measured in 7 volunteers at 7T. METHODS: [Formula: see text] method uses two partially relaxed saturation transfer (ST) spectra and correction factor to determine CK rate constant. Correction factor is determined by numerical simulation of Bloch McConnell equations using known spin and experimental parameters. Optimal parameters and error estimate in calculation of CK reaction rate constant were determined by simulations. The technique was validated in calf muscles by direct comparison with saturation transfer measurements. [Formula: see text] pulse sequence was incorporated with 1D-image selected in vivo spectroscopy, combined with 2D-chemical shift spectroscopic imaging (1D-ISIS/2D-CSI) for studies in heart. The myocardial CK reaction rate constant was then measured in 7 volunteers. RESULTS: Skeletal muscle kf determined by conventional approach and [Formula: see text] approach were the same 0.31 ± 0.02 s-1 and 0.30 ± 0.04 s-1 demonstrating the validity of the technique. Results are reported as mean ± SD. Myocardial CK reaction rate constant was 0.29 ± 0.05 s-1, consistent with previously reported studies. CONCLUSION: [Formula: see text] method enables acquisition of 31P saturation transfer MRS under partially relaxed conditions and enables 2D-CSI of kf in myocardium. This work enables applications for in vivo CSI imaging of energetics in heart and other organs in clinically relevant acquisition time.

Nucleotide Binding Modes in a Motor Protein Revealed by 31 P- and 1 H-Detected MAS Solid-State NMR Spectroscopy.

Protein-nucleic acid interactions play important roles not only in energy-providing reactions, such as ATP hydrolysis, but also in reading, extending, packaging, or repairing genomes. Although they can often be analyzed in detail with X-ray crystallography, complementary methods are needed to visualize them in complexes, which are not crystalline. Here, we show how solid-state NMR spectroscopy can detect and classify protein-nucleic interactions through site-specific 1 H- and 31 P-detected spectroscopic methods. The sensitivity of 1 H chemical-shift values on noncovalent interactions involved in these molecular recognition processes is exploited allowing us to probe directly the chemical bonding state, an information, which is not directly accessible from an X-ray structure. We show that these methods can characterize interactions in easy-to-prepare sediments of the 708 kDa dodecameric DnaB helicase in complex with ADP:AlF4- :DNA, and this despite the very challenging size of the complex.

Pilot study of homeostatic alterations of mineral elements in serum of patients with age-related macular degeneration via elemental and isotopic analysis using ICP-mass spectrometry.

Age-related macular degeneration (AMD), the main cause of irreversible blindness in people over 60 years of age, is an eye disease that evolves with loss of central vision. Although AMD manifests itself in the eye, blood is continuously flowing through the macular region, such that potential alterations in this region could be reflected in the composition of whole blood or plasma/serum. Therefore, the potential clinical relevance of analysis of serum samples was assessed because of the low degree of invasiveness of blood sampling. 40 initial samples (20 from controls and 20 from patients with the dry form of AMD) have been analysed in this work to investigate the possible occurrence of homeostatic alterations of essential mineral elements caused by the disease. Both major (Na, Mg, P and K) and trace (Fe, Cu and Zn) essential mineral elements were determined in blood serum using single-collector ICP-mass spectrometry. Also, the isotopic composition of Cu (an element proposed to be directly involved in the onset of AMD) was determined using multi-collector ICP-mass spectrometry. Unexpected light Cu isotopic compositions in three individuals assumed as controls, resulted in a re-evaluation of their clinical information and a later exclusion due to pathologies initially not accounted for. In this pilot study, a significant alteration in the δ65Cu value has been found between the two final cohorts (AMD patients: n = 20; controls n = 17), with lower δ65Cu values (i.e. an enrichment in the light 63Cu isotope) in the case of AMD. Also, higher serum concentrations of the elements P and Zn were established in AMD at a systemic level.

Fast and slow biomembrane solubilizing detergents: Insights into their mechanism of action.

Detergents are water-soluble amphiphiles. Above a critical concentration they self-organize in micelles and in the presence of phospholipids mixed micelles are formed. Much information is available on the structure of these self-assemblies and on the thermodynamics of their formation. The aim of this study was to deepen our understanding of the mechanisms of solubilization. Solubilization of lipid vesicles made of egg phosphatidylcholine (PC) by twenty one commercially available, structurally heterogeneous detergents, has been assessed by a decrease in turbidity of the vesicle suspension. Both steady-state and time-resolved measurements have been performed. The results show that the detergents under study fall into one of two categories, namely fast-solubilizing and slow-solubilizing detergents. This categorization is independent of detergent concentration, i.e. a "slow" cannot be converted into a "fast" surfactant by increasing its bulk concentration. 31P-NMR spectra indicate that slow-acting detergents cause either a gradual, monotonic micellization of bilayers (sodium dodecyl sulphate), or formation of more complex, perhaps non-lamellar, non-micellar intermediates (dodecylmaltoside). In contrast, fast detergents (e.g. Triton X-100) cause lysis and reassembly of vesicles before bulk solubilization takes place. These results support the idea that membrane solubilization by detergents is rapid only when surfactant transbilayer (flipping) motion is easy.

Neural-network classification of cardiac disease from 31P cardiovascular magnetic resonance spectroscopy measures of creatine kinase energy metabolism.

BACKGROUND: The heart's energy demand per gram of tissue is the body's highest and creatine kinase (CK) metabolism, its primary energy reserve, is compromised in common heart diseases. Here, neural-network analysis is used to test whether noninvasive phosphorus (31P) cardiovascular magnetic resonance spectroscopy (CMRS) measurements of cardiac adenosine triphosphate (ATP) energy, phosphocreatine (PCr), the first-order CK reaction rate kf, and the rate of ATP synthesis through CK (CK flux), can predict specific human heart disease and clinical severity. METHODS: The data comprised the extant 178 complete sets of PCr and ATP concentrations, kf, and CK flux data from human CMRS studies performed on clinical 1.5 and 3 Tesla scanners. Healthy subjects and patients with nonischemic cardiomyopathy, dilated (DCM) or hypertrophic disease, New York Heart Association (NYHA) class I-IV heart failure (HF), or with anterior myocardial infarction are included. Three-layer neural-networks were created to classify disease and to differentiate DCM, hypertrophy and clinical NYHA class in HF patients using leave-one-out training. Network performance was assessed using 'confusion matrices' and 'area-under-the-curve' (AUC) analyses of 'receiver operating curves'. Possible methodological bias and network imbalance were tested by segregating 1.5 and 3 Tesla data, and by data augmentation by random interpolation of nearest neighbors, respectively. RESULTS: The network differentiated healthy, HF and non-HF cardiac disease with an overall accuracy of 84% and AUC > 90% for each category using the four CK metabolic parameters, alone. HF patients with DCM, hypertrophy, and different NYHA severity were differentiated with ~ 80% overall accuracy independent of CMRS methodology. CONCLUSIONS: While sample-size was limited in some sub-classes, a neural network classifier applied to noninvasive cardiac 31P CMRS data, could serve as a metabolic biomarker for common disease types and HF severity with clinically-relevant accuracy. Moreover, the network's ability to individually classify disease and HF severity using CK metabolism alone, implies an intimate relationship between CK metabolism and disease, with subtle underlying phenotypic differences that enable their differentiation. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00181259.

Cerebral bioenergetic differences measured by phosphorus-31 magnetic resonance spectroscopy between bipolar disorder and healthy subjects living in two different regions suggesting possible effects of altitude.

AIM: Increased oxidative stress in cerebral mitochondria may follow exposure to the systemic hypobaric hypoxia associated with residing at higher altitudes. Because mitochondrial dysfunction is implicated in bipolar disorder (BD) pathophysiology, this may impact the cerebral bioenergetics in BD. In this study, we evaluated the cerebral bioenergetics of BD and healthy control (HC) subjects at two sites, located at sea level and at moderate altitude. METHODS: Forty-three veterans with BD and 33 HC veterans were recruited in Boston (n = 22) and Salt Lake City (SLC; n = 54). Levels of phosphocreatine, ß nucleoside triphosphate (ßNTP), inorganic phosphate, and pH over total phosphate (TP) were measured using phosphorus-31 magnetic resonance spectroscopy in the following brain regions: anterior cingulate cortex and posterior occipital cortex, as well as bilateral prefrontal and occipitoparietal (OP) white matter (WM). RESULTS: A significant main effect of site was found in ßNTP/TP (Boston > SLC) and phosphocreatine/TP (Boston < SLC) in most cortical and WM regions, and inorganic phosphate/TP (Boston < SLC) in OP regions. A main effect analysis of BD diagnosis demonstrated a lower pH in posterior occipital cortex and right OP WM and a lower ßNTP/TP in right prefrontal WM in BD subjects, compared to HC subjects. CONCLUSION: The study showed that there were cerebral bioenergetic differences in both BD and HC veteran participants at two different sites, which may be partly explained by altitude difference. Future studies are needed to replicate these results in order to elucidate the dysfunctional mitochondrial changes that occur in response to hypobaric hypoxia.

Measuring inorganic phosphate and intracellular pH in the healthy and hypertrophic cardiomyopathy hearts by in vivo 7T 31P-cardiovascular magnetic resonance spectroscopy.

BACKGROUND: Cardiovascular phosphorus MR spectroscopy (31P-CMRS) is a powerful tool for probing energetics in the human heart, through quantification of phosphocreatine (PCr) to adenosine triphosphate (ATP) ratio. In principle, 31P-CMRS can also measure cardiac intracellular pH (pHi) and the free energy of ATP hydrolysis (ΔGATP). However, these require determination of the inorganic phosphate (Pi) signal frequency and amplitude that are currently not robustly accessible because blood signals often obscure the Pi resonance. Typical cardiac 31P-CMRS protocols use low (e.g. 30°) flip-angles and short repetition time (TR) to maximise signal-to-noise ratio (SNR) within hardware limits. Unfortunately, this causes saturation of Pi with negligible saturation of the flowing blood pool. We aimed to show that an adiabatic 90° excitation, long-TR, 7T 31P-CMRS protocol will reverse this balance, allowing robust cardiac pHi measurements in healthy subjects and patients with hypertrophic cardiomyopathy (HCM). METHODS: The cardiac Pi T1 was first measured by the dual TR technique in seven healthy subjects. Next, ten healthy subjects and three HCM patients were scanned with 7T 31P-MRS using long (6 s) TR protocol and adiabatic excitation. Spectra were fitted for cardiac metabolites including Pi. RESULTS: The measured Pi T1 was 5.0 ± 0.3 s in myocardium and 6.4 ± 0.6 s in skeletal muscle. Myocardial pH was 7.12 ± 0.04 and Pi/PCr ratio was 0.11 ± 0.02. The coefficients of repeatability were 0.052 for pH and 0.027 for Pi/PCr quantification. The pH in HCM patients did not differ (p = 0.508) from volunteers. However, Pi/PCr was higher (0.24 ± 0.09 vs. 0.11 ± 0.02; p = 0.001); Pi/ATP was higher (0.44 ± 0.14 vs. 0.24 ± 0.05; p = 0.002); and PCr/ATP was lower (1.78 ± 0.07 vs. 2.10 ± 0.20; p = 0.020), in HCM patients, which is in agreement with previous reports. CONCLUSION: A 7T 31P-CMRS protocol with adiabatic 90° excitation and long (6 s) TR gives sufficient SNR for Pi and low enough blood signal to permit robust quantification of cardiac Pi and hence pHi. Pi was detectable in every subject scanned for this study, both in healthy subjects and HCM patients. Cardiac pHi was unchanged in HCM patients, but both Pi/PCr and Pi/ATP increased that indicate an energetic impairment in HCM. This work provides a robust technique to quantify cardiac Pi and pHi.

Feasibility of absolute quantification for 31 P MRS at 7 T.

PURPOSE: Phosphorus spectroscopy can differentiate among liver disease stages and types. To quantify absolute concentrations of phosphorus metabolites, sensitivity calibration and transmit field ( B1+ ) correction are required. The trend toward ultrahigh fields (7 T) and the use of multichannel RF coils makes this ever more challenging. We investigated the constraints on reference phantoms, and implemented techniques for the absolute quantification of human liver phosphorus spectra acquired using a 10-cm loop and a 16-channel array at 7 T. METHODS: The effect of phantom conductivity was assessed at 25.8 MHz (1.5 T), 49.9 MHz (3 T), and 120.3 MHz (7 T) by electromagnetic modeling. Radiofrequency field maps ( B1± ) were measured in phosphate phantoms (18 mM and 40 mM) at 7 T. These maps were used to assess the correction of 4 phantom 3D-CSI data sets using 3 techniques: phantom replacement, explicit normalization, and simplified normalization. In vivo liver spectra acquired with a 10-cm loop were corrected with all 3 methods. Simplified normalization was applied to in vivo 16-channel array data sets. RESULTS: Simulations show that quantification errors of less than 3% are achievable using a uniform electrolyte phantom with a conductivity of 0.23-0.86 S.m-1 at 1.5 T, 0.39-0.58 S.m-1 at 3 T, and 0.34-0.42 S.m-1 (16-19 mM KH2 PO4(aq) ) at 7 T. The mean γ-ATP concentration quantified in vivo at 7 T was 1.39 ± 0.30 mmol.L-1 to 1.71 ± 0.35 mmol.L-1 wet tissue for the 10-cm loop and 1.88 ± 0.25 mmol.L-1 wet tissue for the array. CONCLUSION: It is essential to select a calibration phantom with appropriate conductivity for quantitative phosphorus spectroscopy at 7 T. Using an 18-mM phosphate phantom and simplified normalization, human liver phosphate metabolite concentrations were successfully quantified at 7 T.

Dynamic 31 P spectroscopic imaging of skeletal muscles combining flyback echo-planar spectroscopic imaging and compressed sensing.

PURPOSE: Dynamic phosphorus MR spectroscopic imaging (31 P-MRSI) experiments require temporal resolution on the order of seconds to concurrently assess different muscle groups. A highly accelerated pulse sequence combining flyback echo-planar spectroscopic imaging (EPSI) and compressed sensing was developed and tested in a phantom and healthy humans during an exercise-recovery challenge of the lower leg muscles, using a clinical 3T MRI. METHODS: A flyback EPSI readout designed to achieve 2.25 × 2.25 cm2 resolution over a 18 × 18 cm2 field of view (i.e., 8 × 8 matrix) was combined with compressed sensing through the inclusion of pseudorandom gradient blips to sub-sample the ky-kt dimensions by a factor of 2.7×, achieving a temporal resolution of 9 s. The sequence was first tested in a phantom to assess performance compared to fully sampled EPSI (fidEPSI) and phase encoded chemical shift imaging (fidCSI). Then, tests were performed in 11 healthy volunteers during an exercise-recovery challenge of the lower leg muscles. Voxels containing tissue from different muscle groups were evaluated measuring percentage phosphocreatine (%PCr) depletion, time constant of PCr recovery (τPCr) and intracellular pH at rest and following exercise. RESULTS: The sequence was capable to track the dynamic PCr response of multiple muscles simultaneously. No statistical differences were found in the metabolite ratio, pH or linewidth when compared with fidEPSI and fidCSI in the phantom study. Dynamic experiments showed differences in PCr depletion when comparing soleus with gastrocnemius muscles. Intracellular pH, τPCr and %PCr decrease were consistent with reported values. CONCLUSION: Highly accelerated 31 P-MRSI combining flyback EPSI and compressed sensing is capable of assessing concurrent energy metabolism in multiple muscle groups using a clinical 3T MR system.

Modular 31 P wideband inversion transfer for integrative analysis of adenosine triphosphate metabolism, T1 relaxation and molecular dynamics in skeletal muscle at 7T.

PURPOSE: For efficient and integrative analysis of de novo adenosine triphosphate (ATP) synthesis, creatine-kinase-mediated ATP synthesis, T1 relaxation time, and ATP molecular motion dynamics in human skeletal muscle at rest. METHODS: Four inversion-transfer modules differing in center inversion frequency were combined to generate amplified magnetization transfer (MT) effects in targeted MT pathways, including Pi ↔ γ-ATP, PCr ↔ γ-ATP, and 31 Pγ(α)ATP ↔ 31 PßATP . MT effects from both forward and reverse exchange kinetic pathways were acquired to reduce potential bias and confounding factors in integrated data analysis. RESULTS: Kinetic data collected using 4 wideband inversion modules (8 minutes each) yielded the forward exchange rate constants, kPCr→γATP = 0.31 ± 0.05 s-1 and kPi→γATP = 0.064 ± 0.012 s-1 , and the reverse exchange rate constants, kγATP→Pi = 0.034 ± 0.006 s-1 and kγATP→PCr = 1.37 ± 0.22 s-1 , respectively. The cross-relaxation rate constant, σγ(α) ↔ ßATP was -0.20 ± 0.03 s-1 , corresponding to ATP rotational correlation time τc of 0.8 ± 0.1 × 10-7 seconds. The intrinsic T1 relaxation times were Pi (9.2 ± 1.4 seconds), PCr (6.2 ± 0.4 seconds), γ-ATP (1.8 ± 0.1 seconds), α-ATP (1.4 ± 0.1 seconds), and ß-ATP (1.1 ± 0.1 seconds). Muscle ATP T1 values were found to be significantly longer than those previously measured in the brain using a similar method. CONCLUSION: A combination of multiple inversion transfer modules provides a comprehensive and integrated analysis of ATP metabolism and molecular motion dynamics. This relatively fast technique could be potentially useful for studying metabolic disorders in skeletal muscle.