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.

Unveiling a hidden 31 P signal coresonating with extracellular inorganic phosphate by outer-volume-suppression and localized 31 P MRS in the human brain at 7T.

PURPOSE: The study was undertaken to demonstrate that there is more than 1 component in the extracellular Pi31 P signal ( Piex) acquired from human head using nonlocalized 31 P MRS. METHODS: Outer-volume-suppression (OVS) saturation and 1D/2D 31 P CSI were utilized to reveal the presence of an additional component in the Piex signal. RESULTS: 67% of the head extracellular Pi signal was attenuated upon OVS saturation of the peripheral meningeal tissues, likely reflecting elimination of the Pi signal in the meningeal fluids (the blood and CSF). Localized 1D/2D CSI data provided further support for this assignment. Upon correction for the meningeal contribution, the extracellular Pi concentration was 0.51 ± 0.07 mM, whereas the intracellular Pi was 0.85 ± 0.10 mM. The extracellular pH was measured as 7.32 ± 0.04 when using OVS, as compared to 7.39 ± 0.03 when measured without OVS (N = 7 subjects). CONCLUSION: The extracellular Pi signal acquired from the human head using nonlocalized 31 P MRS contains a significant component likely contributed by peripheral blood and CSF in meninges that must be removed in order to use this signal as an endogenous probe for measuring extracellular pH and other properties in the brain.

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.

Adiabatic excitation for 31 P MR spectroscopy in the human heart at 7 T: A feasibility study.

PURPOSE: Phosphorus magnetic resonance spectroscopy (31 P-MRS) provides a unique tool for assessing cardiac energy metabolism, often quantified using the phosphocreatine (PCr)/adenosine triphosphate (ATP) ratio. Surface coils are typically used for excitation for 31 P-MRS, but they create an inhomogeneous excitation field across the myocardium, producing undesirable, spatially varying partial saturation. Therefore, we implemented adiabatic excitation in a 3D chemical shift imaging (CSI) sequence for cardiac 31 P-MRS at 7 Tesla (T). METHODS: We optimized an adiabatic half passage pulse with bandwidth sufficient to excite PCr and γ-ATP together. In addition, the CSI sequence was modified to allow interleaved excitation of PCr and γ-ATP, then 2,3-DPG, to enable PCr/ATP determination with blood correction. Nine volunteers were scanned at 2 transmit voltages to confirm that measured PCr/ATP was independent of B1+ (i.e. over the adiabatic threshold). Six septal voxels were evaluated for each volunteer. RESULTS: Phantom experiments showed that adiabatic excitation can be reached at the depth of the heart using our pulse. The mean evaluated cardiac PCr/ATP ratio from all 9 volunteers corrected for blood signal was 2.14 ± 0.16. Comparing the two acquisitions with different voltages resulted in a minimal mean difference of -0.005. CONCLUSION: Adiabatic excitation is possible in the human heart at 7 T, and gives consistent PCr/ATP ratios. Magn Reson Med 78:1667-1673, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Using solid 13C NMR coupled with solution 31P NMR spectroscopy to investigate molecular species and lability of organic carbon and phosphorus from aquatic plants in Tai Lake, China.

Forms and labilities of plant-derived organic matters (OMs) including carbon (C) and phosphorus (P) were fundamental for understanding their release, degradation and environmental behaviour in lake ecosystems. Thus, solid 13C and solution 31P nuclear magnetic resonance (NMR) spectroscopy were used to characterize biomass of six aquatic plants in Tai Lake, China. The results showed that carbohydrates (61.2% of the total C) were predominant C functional group in the solid 13C NMR spectra of plant biomass, which may indicate high lability and bioavailability of aquatic plants-derived organic matter in lakes. There was 72.6-103.7% of the total P in aquatic plant biomass extracted by NaOH-EDTA extracts. Solution 31P NMR analysis of these NaOH-EDTA extracts further identified several molecular species of P including orthophosphate (50.1%), orthophosphate monoesters (46.8%), DNA (1.6%) and pyrophosphate (1.4%). Orthophosphate monoesters included ß-glycerophosphate (17.7%), hydrolysis products of RNA (11.7%), α-glycerophosphate (9.2%) and other unknown monoesters (2.1%). Additionally, phytate, the major form of organic P in many lake sediments, was detected in floating plant water poppy. These inorganic P (e.g. orthophosphate and pyrophosphate) and organic P (e.g. diester and its degradation products) identified in plant biomass were all labile and bioavailable P, which would play an important role in recycling of P in lakes. These results increased knowledge of chemical composition and bioavailability of OMs derived from aquatic plants in lakes.

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.

Options of partners improve carbon for phosphorus trade in the arbuscular mycorrhizal mutualism.

The mutualism between plants and arbuscular mycorrhizal fungi (AMF) is widespread and has persisted for over 400 million years. Although this mutualism depends on fair resource exchange between plants and fungi, inequality exists among partners despite mechanisms that regulate trade. Here, we use (33) P and (14) C isotopes and a split-root system to test for preferential allocation and reciprocal rewards in the plant-AMF symbiosis by presenting a plant with two AMF that differ in cooperativeness. We found that plants received more (33) P from less cooperative AMF in the presence of another AMF species. This increase in (33) P resulted in a reduced (14) C cost per unit of (33) P from less cooperative AMF when alternative options were available. Our results indicate that AMF diversity promotes cooperation between plants and AMF, which may be an important mechanism maintaining the evolutionary persistence of and diversity within the plant-AMF mutualism.

A multi-stable isotope framework to understand eutrophication in aquatic ecosystems.

Eutrophication is a globally significant challenge facing aquatic ecosystems, associated with human induced enrichment of these ecosystems with nitrogen (N) and phosphorus (P). However, the limited availability of inherent labels for P and N has constrained understanding of the triggers for eutrophication in natural ecosystems and appropriate targeting of management responses. This paper proposes and evaluates a new multi-stable isotope framework that offers inherent labels to track biogeochemical reactions governing both P and N in natural ecosystems. The framework couples highly novel analysis of the oxygen isotope composition of phosphate (δ(18)OPO4) with dual isotope analysis of oxygen and N within nitrate (δ(15)NNO3, δ(18)ONO3) and with stable N isotope analysis in ammonium (δ(15)NNH4). The River Beult in England is used as an exemplar system for initial evaluation of this framework. Our data demonstrate the potential to use stable isotope labels to track the input and downstream fate of nutrients from point sources, on the basis of isotopic differentiation for both P and N between river water and waste water treatment work effluent (mean difference = +1.7‰ for δ(18)OPO4; +15.5‰ for δ(15)NNH4 (under high flow); +7.3‰ for δ(18)ONO3 and +4.4‰ for δ(15)NNO3). Stable isotope data reveal nutrient inputs to the river upstream of the waste water treatment works that are consistent with partially denitrified sewage or livestock sources of nitrate (δ(15)NNO3 range = +11.5 to +13.1‰) and with agricultural sources of phosphate (δ(18)OPO4 range = +16.6 to +19.0‰). The importance of abiotic and metabolic processes for the in-river fate of N and P are also explored through the stable isotope framework. Microbial uptake of ammonium to meet metabolic demand for N is suggested by substantial enrichment of δ(15)NNH4 (by 10.2‰ over a 100 m reach) under summer low flow conditions. Whilst the concentration of both nitrate and phosphate decreased substantially along the same reach, the stable isotope composition of these ions did not vary significantly, indicating that concentration changes are likely driven by abiotic processes of dilution or sorption. The in-river stable isotope composition and the concentration of P and N were also largely constant downstream of the waste water treatment works, indicating that effluent-derived nutrients were not strongly coupled to metabolism along this in-river transect. Combined with in-situ and laboratory hydrochemical data, we believe that a multi-stable isotope framework represents a powerful approach for understanding and managing eutrophication in natural aquatic ecosystems.

Organic matter remineralization predominates phosphorus cycling in the mid-Bay sediments in the Chesapeake Bay.

Chesapeake Bay, the largest and most productive estuary in the U.S., suffers from varying degrees of water quality issues fueled by both point and nonpoint nutrient sources. Restoration of the Bay is complicated by the multitude of nutrient sources, their variable inputs, and complex interaction between imported and regenerated nutrients. These complexities not only restrict formulation of effective restoration plans but also open up debates on accountability issues with nutrient loading. A detailed understanding of sediment phosphorus (P) dynamics provides information useful in identifying the exchange of dissolved constituents across the sediment-water interface as well as helps to better constrain the mechanisms and processes controlling the coupling between sediments and the overlying waters. Here we used phosphate oxygen isotope ratios (δ(18)O(P)) in concert with sediment chemistry, X-ray diffraction, and Mössbauer spectroscopy on sediments retrieved from an organic rich, sulfidic site in the mesohaline portion of the mid-Bay to identify sources and pathway of sedimentary P cycling and to infer potential feedbacks on bottom water hypoxia and surface water eutrophication. Authigenic phosphate isotope data suggest that the regeneration of inorganic P from organic matter degradation (remineralization) is the predominant, if not sole, pathway for authigenic P precipitation in the mid-Bay sediments. This indicates that the excess inorganic P generated by remineralization should have overwhelmed any pore water and/or bottom water because only a fraction of this precipitates as authigenic P. This is the first research that identifies the predominance of remineralization pathway and recycling of P within the Chesapeake Bay. Therefore, these results have significant implications on the current understanding of sediment P cycling and P exchange across the sediment-water interface in the Bay, particularly in terms of the sources and pathways of P that sustain hypoxia and may potentially support phytoplankton growth in the surface water.

Accurate measurement of heteronuclear dipolar couplings by phase-alternating R-symmetry (PARS) sequences in magic angle spinning NMR spectroscopy.

We report a Phase-Alternating R-Symmetry (PARS) dipolar recoupling scheme for accurate measurement of heteronuclear (1)H-X (X = (13)C, (15)N, (31)P, etc.) dipolar couplings in MAS NMR experiments. It is an improvement of conventional C- and R-symmetry type DIPSHIFT experiments where, in addition to the dipolar interaction, the (1)H CSA interaction persists and thereby introduces considerable errors in the dipolar measurements. In PARS, phase-shifted RN symmetry pulse blocks applied on the (1)H spins combined with π pulses applied on the X spins at the end of each RN block efficiently suppress the effect from (1)H chemical shift anisotropy, while keeping the (1)H-X dipolar couplings intact. Another advantage over conventional DIPSHIFT experiments, which require the signal to be detected in the form of a reduced-intensity Hahn echo, is that the series of π pulses refocuses the X chemical shift and avoids the necessity of echo formation. PARS permits determination of accurate dipolar couplings in a single experiment; it is suitable for a wide range of MAS conditions including both slow and fast MAS frequencies; and it assures dipolar truncation from the remote protons. The performance of PARS is tested on two model systems, [(15)N]-N-acetyl-valine and [U-(13)C,(15)N]-N-formyl-Met-Leu-Phe tripeptide. The application of PARS for site-resolved measurement of accurate (1)H-(15)N dipolar couplings in the context of 3D experiments is presented on U-(13)C,(15)N-enriched dynein light chain protein LC8.

In vivo ³¹P-nuclear magnetic resonance studies of glyphosate uptake, vacuolar sequestration, and tonoplast pump activity in glyphosate-resistant horseweed.

Horseweed (Conyza canadensis) is considered a significant glyphosate-resistant (GR) weed in agriculture, spreading to 21 states in the United States and now found globally on five continents. This laboratory previously reported rapid vacuolar sequestration of glyphosate as the mechanism of resistance in GR horseweed. The observation of vacuole sequestration is consistent with the existence of a tonoplast-bound transporter. (31)P-Nuclear magnetic resonance experiments performed in vivo with GR horseweed leaf tissue show that glyphosate entry into the plant cell (cytosolic compartment) is (1) first order in extracellular glyphosate concentration, independent of pH and dependent upon ATP; (2) competitively inhibited by alternative substrates (aminomethyl phosphonate [AMPA] and N-methyl glyphosate [NMG]), which themselves enter the plant cell; and (3) blocked by vanadate, a known inhibitor/blocker of ATP-dependent transporters. Vacuole sequestration of glyphosate is (1) first order in cytosolic glyphosate concentration and dependent upon ATP; (2) competitively inhibited by alternative substrates (AMPA and NMG), which themselves enter the plant vacuole; and (3) saturable. (31)P-Nuclear magnetic resonance findings with GR horseweed are consistent with the active transport of glyphosate and alternative substrates (AMPA and NMG) across the plasma membrane and tonoplast in a manner characteristic of ATP-binding cassette transporters, similar to those that have been identified in mammalian cells.

[Characterization and optimization of the NaOH-EDTA extracts for solution 31P-NMR analysis of organic phosphorus in river sediments].

Optimization and mechanism of NaOH-EDTA extraction solutions were studied in phosphorus (P) pollution river sediments, which were Fe, Al-rich sediment, by solution 31P nuclear magnetic resonance spectroscopy (31P-NMR). Different proportions of NaOH and EDTA showed different extraction efficiency on total P (TP) and organic P (Po) in the sediment. The concentration of Po in NaOH + EDTA extract was higher than that in NaOH extract. The mechanism was that the TP and Po were released under the conditions of EDTA chelating with Fe and Al. The concentration of TP and Po were the highest in 1.00 mol x L(-1) NaOH +75 mmol x L(-1) EDTA extract and 0.25 mol x L(-1) NaOH + 50 mmol x L(-1) EDTA extract, which were 3.88 mg x g(-1) and 0.24 mg x g(-1), respectively. The extractions of Fe, Mn, Ca, Mg, Al were increasing as the EDTA increased under the same NaOH concentration. Extraction efficiency of Fe, Mn, Ca showed negative correlation with the pH of the extracting solution (P < 0.01). Exponential relationship was found between the extraction of Al and the pH of the extraction solution (P < 0.01) because of the AlO2- and EDTA-Al complex. The quality of spectra of NaOH-EDTA extract was better than that of NaOH extract. Six P species were detected in different extractions, including phosphonates, orthophosphate, pyrophosphate, orthophosphate monoesters, phospholipids and deoxyribonucleic acids. Therefore, 0. 25 mol x L(-1) NaOH + 50 mmol x L(-1) EDTA was the optimization extraction solution for Po analysis in Fe and Al-rich river sediment by 31P-NMR.

Comparison of three reference methods for the measurement of intracellular pH using 31P MRS in healthy volunteers and patients with lymphoma.

31P magnetic resonance spectroscopy (31P MRS) can measure intracellular pH (pHi) using the chemical shift difference between pH-dependent inorganic phosphate (Pi) and a pH-independent reference peak. This study compared three different frequency reference peaks [phosphocreatine (PCr), α resonance of adenosine triphosphate (αATP) and water (using 1H MRS)] in a cohort of 10 volunteers and eight patients with non-Hodgkin's lymphoma (NHL). Well-resolved chemical shift imaging (CSI) spectra were acquired on a 1.5T scanner for muscle, liver and tumour. The pH was calculated for all volunteers and patients using the available methods. The consistency of the resulting pH was evaluated. The direct Pi­PCr method was best for those spectra with a very well-defined PCr, such as muscle (pH=7.05 ± 0.02). In liver, the Pi­αATP method gave more consistent results (pH=7.30 ± 0.06) than the calibrated water-based method (pH=7.27 ± 0.11). In NHL nodes, the measured pH using the Pi­αATP method was 7.25 ± 0.12. Given that the measured range includes some biological variation in individual patients, treatment-related changes of the order of 0.1 pH units should be detectable.

Noninvasive phosphorus magnetic resonance spectroscopic imaging predicts outcome to first-line chemotherapy in newly diagnosed patients with diffuse large B-cell lymphoma.

RATIONALE AND OBJECTIVES: Based on their association with malignant proliferation, using noninvasive phosphorus MR spectroscopic imaging ((31)P MRSI), we measured the tumor content of the phospholipid-related phosphomonoesters (PME), phosphoethanolamine and phospholcholine, and its correlation with treatment outcome in newly diagnosed patients with diffuse large B-cell lymphoma (DLBCL) receiving standard first-line chemotherapy. EXPERIMENTAL DESIGN: The PME value normalized to nucleoside triphosphates (PME/NTP) was measured using (31)P MRSI in tumor masses of 20 patients with DLBCL before receiving standard first-line chemotherapy. Response at 6 months was complete in 13 patients and partial in seven. Time to treatment failure (TTF) was ≤11 months in eight patients, from 18 to 30 months in three, and ≥60 months in nine. RESULTS: On a t test, the pretreatment tumor PME/NTP mean value (SD, n) of patients with a complete response at 6 months was 1.42 (0.41, 13), which was significantly different from the value of 2.46 (0.40, 7) in patients with partial response (P < .00001). A Fisher test significantly correlated the PME/NTP values with response at 6 months (sensitivity and specificity at 0.85, P < .004) while a Cox proportional hazards regression significantly correlated the PME/NTP values with TTF (hazard ratio = 5.21, P < .02). A Kaplan-Meier test set apart a group entirely composed of patients with TTF ≤ 11 months (hazard ratio = 8.66, P < .00001). CONCLUSIONS: The pretreatment tumor PME/NTP values correlated with response to treatment at 6 months and time to treatment failure in newly diagnosed patients with DLBCL treated with first-line chemotherapy, and therefore they could be used to predict treatment outcome in these patients.

A modified protocol for retrograde cerebral perfusion: magnetic resonance spectroscopy in pigs.

OBJECTIVES: Retrograde cerebral perfusion (RCP) has been employed to protect the brain during cardiovascular surgery, requiring temporary hypothermic circulatory arrest (HCA). However, the protocol used for RCP remains to be modified if prolonged HCA is expected. The aim of this study was to determine the efficacy of a modified protocol for this purpose. METHODS: After establishment of HCA at 15°C, 14 pigs were subjected to 90-min RCP using either the conventional protocol (i.e. alpha-stat strategy, 25-mmHg perfusion pressure and occluded inferior vena cava, Group I, n = 7) or the new protocol (i.e. pH-stat strategy, 40-mmHg perfusion pressure and unoccluded inferior vena cava, Group II, n = 7). After being rewarmed to 37°C, pigs were perfused for another 60 min. Phosphorus-31 magnetic resonance spectroscopy was used to track the changes of brain high-energy phosphates [i.e. adenosine triphosphate and phosphocreatine (PCr)] and intracellular pH (pHi). At the end, brain water content was measured. RESULTS: During RCP, high-energy phosphates decreased in both groups, whereas adenosine triphosphate decreased much faster in Group I (10.4 ± 4.3 vs 30.4 ± 4.4% of the baseline, P = 0.007, 60-min RCP). After rewarming, the recovery of high-energy phosphates and pHi was much slower in Group I (PCr: 55.7 ± 9.1 vs 78.4 ± 5.1% of the baseline, P = 0.046; adenosine triphosphate: 26.6 ± 10.6 vs 64.8 ± 4.6% of the baseline, P = 0.007; pHi: 6.5 ± 0.4 vs 7.1 ± 0.1, P = 0.021 at 30-min normothermic perfusion after rewarming). Brain tissue water content was significantly higher in Group I (81.1 ± 0.4 vs 79.5 ± 0.4%, P = 0.016). CONCLUSIONS: Application of the modified RCP protocol significantly improved cerebral energy conservation during HCA and accelerated energy recovery after rewarming.

Enhanced enzymatic thermal stability and activity in functionalized mesoporous silica monitored by (31) p NMR.

Organophosphorus hydrolase (OPH) is immobilized on ammonium-modified mesoporous silica particles. Thermal stability and activity are measured with a (31) P NMR assay of the conversion of paraoxon (toxic) to its non-toxic hydrolysis product. After immobilization, OPH is significantly more active at room temperature and retained activity even after being heated to 45 °C for 1 month.

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.

Mycorrhizal networks: common goods of plants shared under unequal terms of trade.

Plants commonly live in a symbiotic association with arbuscular mycorrhizal fungi (AMF). They invest photosynthetic products to feed their fungal partners, which, in return, provide mineral nutrients foraged in the soil by their intricate hyphal networks. Intriguingly, AMF can link neighboring plants, forming common mycorrhizal networks (CMNs). What are the terms of trade in such CMNs between plants and their shared fungal partners? To address this question, we set up microcosms containing a pair of test plants, interlinked by a CMN of Glomus intraradices or Glomus mosseae. The plants were flax (Linum usitatissimum; a C(3) plant) and sorghum (Sorghum bicolor; a C(4) plant), which display distinctly different (13)C/(12)C isotope compositions. This allowed us to differentially assess the carbon investment of the two plants into the CMN through stable isotope tracing. In parallel, we determined the plants' "return of investment" (i.e. the acquisition of nutrients via CMN) using (15)N and (33)P as tracers. Depending on the AMF species, we found a strong asymmetry in the terms of trade: flax invested little carbon but gained up to 94% of the nitrogen and phosphorus provided by the CMN, which highly facilitated growth, whereas the neighboring sorghum invested massive amounts of carbon with little return but was barely affected in growth. Overall biomass production in the mixed culture surpassed the mean of the two monocultures. Thus, CMNs may contribute to interplant facilitation and the productivity boosts often found with intercropping compared with conventional monocropping.

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.

Selective detection of peptide-oligonucleotide heteroconjugates utilizing capillary HPLC-ICPMS.

A method for the selective detection and quantification of peptide:oligonucleotide heteroconjugates, such as those generated by protein:nucleic acid cross-links, using capillary reversed-phase high performance liquid chromatography (cap-RPHPLC) coupled with inductively coupled plasma mass spectrometry detection (ICPMS) is described. The selective detection of phosphorus as (31)P(+), the only natural isotope, in peptide-oligonucleotide heteroconjugates is enabled by the elemental detection capabilities of the ICPMS. Mobile phase conditions that allow separation of heteroconjugates while maintaining ICPMS compatibility were investigated. We found that trifluoroacetic acid (TFA) mobile phases, used in conventional peptide separations, and hexafluoroisopropanol/triethylamine (HFIP/TEA) mobile phases, used in conventional oligonucleotide separations, both are compatible with ICPMS and enable heteroconjugate separation. The TFA-based separations yielded limits of detection (LOD) of ~40 ppb phosphorus, which is nearly seven times lower than the LOD for HFIP/TEA-based separations. Using the TFA mobile phase, 1-2 pmol of a model heteroconjugate were routinely separated and detected by this optimized capLC-ICPMS method.