In vivo 13C NMR determines metabolic fluxes and steady state in linseed embryos

The dynamics of developing linseed embryo metabolism was investigated using (13)C-labelling experiments where the real-time kinetics of label incorporation into metabolites was monitored in situ using in vivo NMR. The approach took advantage of the occurrence in this plant tissue of large metabolite pools - such as sucrose or lipids - to provide direct and quantitative measurement of the evolution of the labelling state within central metabolism. As a pre-requisite for the use of steady state flux measurements it was shown that isotopic steady state was reached within 3 h at the level of central intermediates whereas it took a further 6h for the sucrose pool. Complete isotopic and metabolic steady state took 18 h to be reached. The data collected during the transient state where label was equilibrated but the metabolic steady state was incomplete, enabled the rates of lipid and sucrose synthesis to be measured in situ on the same sample. This approach is suitable to get a direct assessment of metabolic time-scales within living plant tissues and provides a valuable complement to steady state flux determinations.     

In vivo 31P and multilabel 13C NMR measurements for evaluation of plant metabolic pathways

Reliable measurements of intracellular metabolites are useful for effective plant metabolic engineering. This study explored the application of in situ 31P and 13C NMR spectroscopy for long-term measurements of intracellular pH and concentrations of several metabolites in glycolysis, glucan synthesis, and central carbon metabolic pathways in plant tissues. An NMR perfusion reactor system was designed to allow Catharanthus roseus hairy root cultures to grow for 3-6 weeks, during which time NMR spectroscopy was performed. Constant cytoplasmic pH (7.40+/-0.06), observed during the entire experiment, indicated adequate oxygenation. 13C NMR spectroscopy was performed on hairy root cultures grown in solutions containing 1-13C-, 2-13C-, and 3-13C-labeled glucose in separate experiments and the flow of label was monitored. Activities of pentose phosphate pathways, nonphotosynthetic CO2 fixation, and glucan synthesis pathways were evident from the experimental results. Scrambling of label in glucans also indicated recycling of triose phosphate and their subsequent conversion to hexose phosphates.     

Continuous-flow NMR culture system for mammalian cells

A continuous-flow NMR culture system for mammalian cells has been developed on which ³¹P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by ³¹P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a ΔpH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.     

In vivo NMR metabolic profiling of Fabrea salina reveals sequential defense mechanisms against ultraviolet radiation

Fabrea salina is a hypersaline ciliate that is known to be among the strongest ultraviolet (UV)-resistant microorganisms; however, the molecular mechanisms of this resistance are almost unknown. By means of in vivo NMR spectroscopy, we determined the metabolic profile of living F. salina cells exposed to visible light and to polychromatic UV-B + UV-A + Vis radiation for several different exposure times. We used unsupervised pattern-recognition analysis to compare these profiles and discovered some metabolites whose concentration changed specifically upon UV exposure and in a dose-dependent manner. This variation was interpreted in terms of a two-phase cell reaction involving at least two different pathways: an early response consisting of degradation processes, followed by a late response activating osmoprotection mechanisms. The first step alters the concentration of formate, acetate, and saturated fatty-acid metabolites, whereas the osmoprotection modifies the activity of betaine moieties and other functionally related metabolites. In the latter pathway, alanine, proline, and sugars suggest a possible incipient protein synthesis as defense and/or degeneration mechanisms. We conclude that NMR spectroscopy on in vivo cells is an optimal approach for investigating the effect of UV-induced stress on the whole metabolome of F. salina because it minimizes the invasiveness of the measurement.     

In vivo metabolic intermediates of phospholipid biosynthesis in Rhodopseudomonas sphaeroides

The in vivo metabolic pathways of phospholipid biosynthesis in Rhodopseudomonas sphaeroides have been investigated. Rapid pulse-chase-labeling studies indicated that phosphatidylethanolamine and phosphatidylglycerol were synthesized as in other eubacteria. The labeling pattern observed for N-acylphosphatidylserine (NAPS) was inconsistent with the synthesis of this phospholipid occurring by direct acylation of phosphatidylserine (PS). Rather, NAPS appeared to be kinetically derived from an earlier intermediate such as phosphatidic acid or more likely CDP-diglyceride. Tris-induced NAPS accumulation specifically reduced the synthesis of PS. Treatment of cells with a bacteriostatic concentration of hydroxylamine (10 mM) greatly reduced total cellular phospholipid synthesis, resulted in accumulation of PS, and stimulated the phosphatidylglycerol branch of phospholipid metabolism relative to the PS branch of the pathway. When the cells were treated with a lower hydroxylamine dosage (50 microM), total phospholipid synthesis lagged as PS accumulated, however, phospholipid synthesis resumed coincident with a reversal of PS accumulation. Hydroxylamine alone was not sufficient to promote NAPS accumulation but this compound allowed continued NAPS accumulation when cells were grown in medium containing Tris. The significance of these observations is discussed in terms of NAPS biosynthesis being representative of a previously undescribed branch of the phospholipid biosynthetic sequence.     

In vivo metabolic imaging of cardiac bioenergetics in transgenic mice

Recent advances in transgenic technology have made the mouse a particularly interesting small animal in cardiovascular research. Increasingly sophisticated experimental methods and tools are needed for detailed characterization of cardiovascular physiology and biochemistry in the mice. The objective of this study was to develop a method for noninvasive evaluation of cardiac energy metabolism in the mouse. Cardiac gated (31)P magnetic resonance spectroscopy using Image Selected in Vivo Spectroscopy (ISIS) method was applied in old mice overexpressing bovine growth hormone (bGH) (n = 5) and control mice (n = 5). The localized volumes of interest were 128 and 112 microL, respectively. Phosphocreatine-to-ATP ratio was 1.5 +/- 0.13 in the bGH mice and 2.1 +/- 0.04 in the control group (P < 0.01). The study demonstrates the feasibility of application of volume-selective (31)P MRS for evaluation of cardiac energy metabolism in the mouse under maintained physiological conditions.     

In vivo functional NMR imaging of resistance artery control

Arterial spin labeling (ASL) in combination with NMR imaging is an in vivo technique that quantifies tissue perfusion in absolute values (ml blood x min(-1) x g tissue(-1)) with high temporal (1-10 s) and spatial (0.1-3 mm) resolution. It uses the arterial water spins as endogenous freely diffusible markers of perfusion and, hence, is a totally noninvasive method. The technique has been successfully applied to quantify baseline perfusion in many organs, including the heart, in humans and animals, and results were validated by comparison with gold standards, PET and microspheres, respectively. Because of the high sampling rate of perfusion with ASL and the possibility that measurements could be obtained without harm over indefinite periods of time, the technique has the potential for use in functional investigations of microcirculation regulation and resistance artery control in vivo. We describe examples of the use of ASL to this end. With use of specific technological developments, ASL determination of perfusion can be coupled with simultaneous acquisitions of (1)H and (31)P NMR spectroscopy data. These protocols offer new possibilities whereby the microcirculatory control of cell oxygenation and high-energy phosphate metabolism can be explored.     

In vivo NMR detection of diet-induced changes in adipose tissue composition

We introduce an in vivo spectroscopic method to assess the effects of diet on fatty acid composition of the predominant chemical constituent of adipocytes in mice. To do this, we make use of a nonlinear NMR signal that, unlike a standard NMR signal, is intrinsically insensitive to local magnetic field inhomogeneities and which naturally suppresses the large water signal from nonfatty tissues. Our method yields fat composition information from fat depots distributed over large sample volumes in a single experiment, without requiring the use of tedious shimming procedures, voxel selection, or water suppression. Our results suggest that this method can reveal clear differences in adipose tissue composition of mice fed a standard chow diet compared with mice fed a diet rich in polyunsaturated fatty acids. With further developments this method could be used to obtain information on human lipid composition noninvasively and to track changes in lipid composition induced by diet intervention, pharmaceutical drugs, and exercise.     

In vivo 19F NMR chemical-shift imaging of Ancistrocladus species

Summary ¹⁹F nuclear magnetic resonance (NMR) imaging and¹⁹F NMR chemical-shift imaging (¹⁹F CSI) have been used to localize fluorinated compounds administered to stems ofAncistrocladus heyneanus andA. abbreviatus for the elucidation of biosynthetic pathways in living plants. This first application of¹⁹F CSI on plants proved CSI to be a valuable technique for mapping fluorinated molecules in plants. Exemplarily using trifluoroacetate as a model compound allowed to select appropriate feeding methods and to optimize both concentration and duration of the application to the plant. The time course of the uptake and distribution of trifluoroacetate was monitored by both¹⁹F imaging and¹⁹F CSI. Fluorinated metabolites formed by uptake of 3-fluoro-3-deoxy-D-glucose were detected with¹⁹F CSI.Abbreviations 3-FDG 3-fluoro-3-deoxy-D-glucose - CSI chemicalshift imaging - NMR nuclear magnetic resonance - SNR signal-to-noise ratio - TFA trifluoroacetate Dedicated to Professor Manfred Christi on the occasion of his 60th birthday     

In vivo skin penetration and metabolic path of quantum dots

The skin is the largest organ of the body and is a potential route of exposure to sunscreens and cosmetics containing nanoparticles;however,the permeability of the skin to these nanoparticles is currently unknown.In this paper,we studied the transdermal delivery capacity through mouse skin of water-soluble CdSeS quantum dots(QDs) and the deposition of these QDs in the body.QD solution was coated onto the dorsal hairless skin of male ICR mice.Fluorescence microscopy and transmission electron microscopy(TEM) were used to observe the distribution of QDs in the skin and organs,and inductively coupled plasma-mass spectrometry(ICP-MS) was used to measure the 111Cd content to indicate the concentration of QDs in plasma and organs.Experimental results indicate that QDs can penetrate into the dermal layer and are limited to the uppermost stratum corneum layers and the hair follicles.Through blood circulation,QDs deposit mostly in liver and kidney and are difficult to clear.111Cd concentration was greater than 14 ng g-1 in kidney after 120 h after 0.32 nmol QDs was applied to a mouse.These results suggest that QDs have in vivo transdermal delivery capacity through mouse skin and are harmful to the liver and kidney.     

Development and application of a membrane cyclone reactor for in vivo NMR spectroscopy with high microbial cell densities

A new bioreactor system has been developed for in vivo NMR spectroscopy of microorganisms under defined physiological conditions. This cyclone reactor with an integrated NMR flow cell is continuously operated in the magnet of a 400-MHz wide-bore NMR spectrometer system. The residence times of medium and cells are decoupled by a circulation-integrated cross-flow microfiltration module to achieve higher cell densities as compared to continuous fermentations without cell retention (increase in cell density up to a factor of 10 in steady state). Volumetric mass transfer coefficients k(L)a of more than 1.0 s(-1) are possible in the membrane cyclone reactor, ensuring adequate oxygen supply [oxygen transfer rate >15,000 mg O(2) .(L h)(-1)] of high cell densities. With the aid of the membrane cyclone reactor we were able to show, using continuous in vivo (31)P NMR spectroscopy of anaerobic glucose fermentation by Zymomonas mobilis, that the NMR signal intensity was directly proportional to the cell concentration in the reactor. The concentration profiles of intracellular inorganic phosphate, NAD(H), NDP, NTP, UDP-sugar, a cyclic pyrophosphate, two sugar phosphate pools, and extracellular inorganic phosphate were recorded after a shift from one steady state to another. The intracellular cyclic pyrophosphate had not been detected before in in vitro measurements of Zymomonas mobilis extracts due to the high instability of this compound. Using continuous in vivo (13)C NMR spectroscopy of aerobic glucose utilization by Corynebacterium glutamicum at a density of 25 g(cell dry weight) . L(-1), the membrane cyclone reactor served to measure the different dynamics of labeling in the carbon atoms of L-lactate, L-glutamate, succinate, and L-lysine with a time resolution of 10 min after impressing a [1-(13)C]-glucose pulse.     

In vivo effect of copper status on cisplatin-induced nephrotoxicity

Cisplatin is a widely used antitumor agent; however, tumor resistance and severe side effects limit its use. It is well accepted that cisplatin toxicity can be modulated in vitro in cell cultures by copper salts. In the present work, mice with different blood serum copper status were treated with a single intraperitoneal cisplatin injection at a dose of 5 mg/kg, monitored for 3 days in metabolic cages and analyzed for renal function. Both copper-deficient and copper-overloaded mice displayed more severe early proteinuria and retarded platinum excretion than control mice. The effects of copper status on cisplatin-induced nephrotoxicity are discussed.     

Method for evaluating metabolic functions of drugs in bioartificial liver

Lidocaine and galactose loading tests were performed on a bioartificial liver (BAL), an extracorporeal medical device incorporating living hepatocytes in a cartridge without a transport barrier across the membranes. The concentration changes were analyzed using pharmacokinetic equations to evaluate the efficacy and limitation of the proposed method. Lidocaine and galactose were found to be suitable drugs for a quantitative evaluation of the BAL functions, as they did not interact with the plasma proteins or blood vessels, making their concentrations easy to determine. The drug concentration changes after drug loading were easily analyzed using pharmacokinetic equations, and the BAL functions quantitatively expressed by pharmacokinetic parameters, such as the clearance (CL) and galactose elimination capacity (GEC). In addition, these two drugs have already been used in clinical tests to evaluate human liver functions over long periods, and lidocaineCL values andGEC values reported for a normal human liver. Thus, a comparison of theCL andGEC values for theBAL and a natural liver revealed what proportion of normal liver functions could be replaced by the BAL.     

Investigation of microbial growth in vivo: Evaluation of a novel in vivo chamber implant system

An intraperitoneal chamber implant system has been used to investigate the phenotype of Staphylococcus aureus growing in the rat and the effect of the antibiotic flucloxacillin on bacterial growth in vivo. Titanium chambers were implanted in the peritoneum: a period of 3-4 days equilibration allowed diffusion of host proteins into the chamber fluid prior to inoculation with bacteria. S. aureus inoculated into the chamber fluid, grew rapidly over a 72 h period, reaching counts of > 10(9) per ml. Organisms harvested from chambers were analysed by SDS-PAGE and showed significant differences in polypeptide profiles from the same strain grown in nutrient broth in vitro. Analysis of whole cell extracts by Western-blotting revealed that protein A expression was repressed in S. aureus grown in vivo. Following subcutaneous administration, flucloxacillin levels in serum peaked earlier and were higher than those detected in chamber fluid. The inhibitory effect of the antibiotic on the growth of S. aureus in chambers in treated animals could be monitored easily by sequential sampling of the chamber fluid. These results indicate the potential of the chamber implant model for investigation of microbial phenotype in vivo and development of alternative methods for assessment of antimicrobial efficacy in vivo.     

In vivo approaches and rationale for quantifying kinetics and imaging brain lipid metabolic pathways

Developing a kinetic strategy to examine rates of lipid metabolic pathways can help to elucidate the roles that lipids play in tissue function and structure in health and disease. This review summarizes such a strategy, and shows how it has been applied to quantify different kinetic aspects of brain lipid metabolism in animals and humans. Methods involve injecting intravenously a radioactive or heavy isotope labeled substrate that will be incorporated into a lipid metabolic pathway, and using chemical analytical and/or imaging procedures (e.g., quantitative autoradiography or positron emission tomography) to determine tracer distribution in brain regions and their lipid compartments as a function of time. From the measurements, fluxes, turnover rates, half-lives and ATP consumption rates can be calculated, and incorporation rates can be imaged. Experimental changes in these kinetic parameters can help to identify changes in the expression of regulatory enzymes, and thus aid in drug targeting. Cases that are discussed are arachidonic acid turnover and imaging of neuroreceptor-initiated phospholipase A2 activation, ether phospholipid biosynthesis, and kinetics of the phosphatidylinositol cycle.     

A tripartite microbial reporter gene system for real-time assays of soil nutrient status

Plant-derived carbon is the substrate which drives the rate of microbial assimilation and turnover of nutrients, in particular N and P, within the rhizosphere. To develop a better understanding of rhizosphere dynamics, a tripartite reporter gene system has been developed. We used three lux-marked Pseudomonas fluorescens strains to report on soil (1) assimilable carbon, (2) N-status, and (3) P-status. In vivo studies using soil water, spiked with C, N and P to simulate rhizosphere conditions, showed that the tripartite reporter system can provide real-time assessment of carbon and nutrient status. Good quantitative agreement for bioluminescence output between reference material and soil water samples was found for the C and P reporters. With regard to soil nitrate, the minimum bioavailable concentration was found to be greater than that analytically detectable in soil water. This is the first time that bioavailable soil C, N and P have been quantified using a tripartite reporter gene system.     

In vivo 13C NMR studies of compartmentalized cerebral carbohydrate metabolism

Localized 13C nuclear magnetic resonance (NMR) spectroscopy provides a unique window for studying cerebral carbohydrate metabolism through, e.g. the completely non-invasive measurement of cerebral glucose and glycogen metabolism. In addition, label incorporation into amino acid neurotransmitters such as glutamate (Glu), GABA and aspartate can be measured providing information on Krebs cycle flux and oxidative metabolism. Given the compartmentation of key enzymes such as pyruvate carboxylase and glutamine synthetase, the detection of label incorporation into glutamine indicated that neuronal and glial metabolism can be measured in vivo. The purpose of this paper is to provide a critical overview of these recent advances into measuring compartmentation of brain energy metabolism using localized in vivo 13C NMR spectroscopy. The studies reviewed herein showed that anaplerosis is significant in brain, as is oxidative ATP generation in glia and the rate of glial glutamine synthesis attributed to the replenishment of the neuronal Glu pool and that brain glycogen metabolism is slow under resting conditions. This new modality promises to provide a new investigative tool to study aspects of normal and diseased brain hitherto unaccessible, such as the interplay between glutamatergic action, glucose and glycogen metabolism during brain activation, and the derangements thereof in patients with hepatic encephalopathy, neurodegenerative diseases and diabetes.