Thomas Schleich

Spatial localization of tissue metabolites by phosphorus-31 NMR rotating-frame zeugmatography

Abstract Surface coils are employed for rotating-frame zeugmatography experiments to accomplish one-dimensional 31 P metabolite mapping in phantoms and bovine eyes. The spatially dependent β 1 field of the surface coil provides the necessary magnetic field gradient, and the experiments are designed to acquire 15–32 spectra obtained with rf pulses of equally incremented durations. Regions of the sample are distinguished on the basis of the differing magnetization precessional rates about the spatially dependent β 1 field. Thus the mapping dimension corresponds to changes in the transverse ( x′y′ ) magnetization components of the β 1 field, which correspond to successive curved surfaces of constant β 1 field strength. Two-dimensional Fourier transform processing of the data results in a metabolite map composed of a suite of spectra displayed as a function of the mapping dimension. Experiments with bovine eye phantoms reveal that T 1 discrimination can produce a loss of resolution due to smearing in the mapping dimension. Resonance offset effects are also apparent at distances removed from the coil. Elimination of the T 1 discrimination-induced smearing is demonstrated through the incorporation of a saturation pulse into the experiment. Application of the method to the intact bovine eye is shown to result in metabolite maps delineating contributions from constituent ocular tissues of the bovine eye.

A modified rotating frame experiment based on a fourier series window function. Application to in vivo spatially localized NMR spectroscopy

A fourier series window approach to the rotating frame experiment is employed in conjunction with surface coils to accomplish one-dimensional spatial localization of phosphorus-containing metabolites in phantoms and the in vivo rat brain. The rotating frame experiment delineates sample regions on the basis of a B1 field gradient across the sample which for the experiments described in this paper are provided by a surface coil. In the usual rotating frame experiment, a suite of spectra (termed a metabolite map) is obtained by incrementing the evolution pulse duration with the spectra displayed as a function of the nutational frequency about the B1 field. The total time of the experiment depends on the desired degree of spatial resolution, and substantial time savings can be obtained when the desired result is spectral information from a single or a few selected regions (slices). The approach taken here is to utilize a Fourier series window function representation of the nutational frequencies over the region of interest. This procedure allows convenient selection of the localized region, thus avoiding Fourier transformation in the second (mapping) dimension. For the case of a single region of interest, the spectral parameters can be adjusted to maximize the number of data files in which the evolution pulse nutates the magnetizations at the center of the slice through a multiple of 180°. Data files not contributing significant signal to a selected slice can be omitted to further minimize the experimental accumulation time.

Incorporation of magnetization transfer into the formalism for rotating-frame spin-lattice proton NMR relaxation in the presence of an off-resonance-irradiation field

Abstract The formalism for off resonance rotating-frame spin-lattice relaxation was extended to include the effects of nuclear-spin magnetization transfer between two sites (A ⇆ B) in which either partial or complete saturation occurs as a consequence of off resonance irradiation and chemical exchange. With the essential features of the classical saturation transfer experiment retained, analytical expressions were derived describing the time-dependent and steady-state behavior of the exchanging nuclear magnetization experiencing off-resonance field-induced partial or complete saturation. Computer simulations are presented illustrating the exchange characteristics of free and G-actin-bound ATP, the phosphoryl exchange between phosphocreatine and the γ-phosphorul position of ATP, and the exchange of phosphorus metabolite between two different Maxation environments, representing mobile and relatively immobile pools of metabolites, in the presence of off resonance irradiation. Theoretical steady-state spectral-intensity-ratio dispersion (variable applied off resonance frequency) curves were found to be sensitive to the values of both the forward and the reverse pseudo-first-order rate constants and the equilibrium constant. Intensity-ratio dispersion curves for slow to intermediate exchange rates showed enhanced separation from one another with increasing off resonance B2 field strength. Application of the chemical-exchange-modified formalism to off resonance saturation data obtained for phosphomonoester metabolites in nuclear bovine calf eye-:lens-tissue homogenates suggests the occurrence of exchange between mobile and immobilized metabolite pools. The formalism derived in this study permits an evaluation of conventional saturation-transfer experiments of slowly tumbling or motionally restricted metabolites where nonselective, or off resonance, partial saturation becomes appreciable. The potential for obtaining motional dynamics and exchange kinetics information from in vivo systems involved in chemical-exchange processes is demonstrated.

Fractionation and structural elucidation of the active components of aurintricarboxylic acid, a potent inhibitor of protein nucleic acid interactions

Commercially available, as well as synthetically prepared, samples of aurintricarboxylic acid (a widely employed potent inhibitor of protein nucleic acid interactions) consist mostly of a heterogeneous collection of polymers, as shown by fractionation schemes utilizing both dialysis and ultrafiltration, and by molecular weight measurements. 13C-NMR studies suggest that the polymeric material is of the phenol-formaldehyde type; inhibitory assays that depend on the formation of a protein-nucleic acid complex revealed that potency varied directly with the molecular weight of the polymer. Fractions of molecular weight 400 were essentially inactive.

Creatine Fails to Augment the Benefits from Resistance Training in Patients with HIV Infection: A Randomized, Double-Blind, Placebo-Controlled Study

Background Progressive resistance exercise training (PRT) improves physical functioning in patients with HIV infection. Creatine supplementation can augment the benefits derived from training in athletes and improve muscle function in patients with muscle wasting. The objective of this study was to determine whether creatine supplementation augments the effects of PRT on muscle strength, energetics, and body composition in HIV-infected patients. Methodology/Principal Findings This is a randomized, double blind, placebo-controlled, clinical research center-based, outpatient study in San Francisco. 40 HIV–positive men (20 creatine, 20 placebo) enrolled in a 14-week study. Subjects were randomly assigned to receive creatine monohydrate or placebo for 14 weeks. Treatment began with a loading dose of 20 g/day or an equivalent number of placebo capsules for 5 days, followed by maintenance dosing of 4.8 g/day or placebo. Beginning at week 2 and continuing to week 14, all subjects underwent thrice-weekly supervised resistance exercise while continuing on the assigned study medication (with repeated 6-week cycles of loading and maintenance). The main outcome measurements included muscle strength (one repetition maximum), energetics (31P magnetic resonance spectroscopy), composition and size (magnetic resonance imaging), as well as total body composition (dual-energy X-ray absorptiometry). Thirty-three subjects completed the study (17 creatine, 16 placebo). Strength increased in all 8 muscle groups studied following PRT, but this increase was not augmented by creatine supplementation (average increase 44 vs. 42%, difference 2%, 95% CI −9.5% to 13.9%) in creatine and placebo, respectively). There were no differences between groups in changes in muscle energetics. Thigh muscle cross-sectional area increased following resistance exercise, with no additive effect of creatine. Lean body mass (LBM) increased to a significantly greater extent with creatine. Conclusions / Significance Resistance exercise improved muscle size, strength and function in HIV-infected men. While creatine supplementation produced a greater increase in LBM, it did not augment the robust increase in strength derived from PRT. Trial Registration ClinicalTrials.gov NCT00484627

Longitudinal (T1) relaxation times of phosphorus metabolites in the bovine and rabbit lens

Longitudinal (T1) magnetic relaxation times for the major phosphorus-containing metabolites present in the bovine and rabbit lens under organ culture conditions and in the bovine and rabbit globe have been determined. Significant differences in T1 for the major phosphorus metabolites in each case are observed, as well as for the same metabolite in the two species examined. Species-dependent lens hydration may account, in part, for these differences. Because of the requirement for rapid repetitive pulsing for the attainment of optimum signal collection efficiency by the Fourier transform nuclear magnetic resonance method, significant differential saturation of metabolite resonance intensities occurs in circumstances where appreciable differences in T1 relaxation times are present, which, unless corrected, leads to erroneous determinations of relative metabolite levels. The net effect of assessing relative metabolite levels in terms of the percentage of total phosphate signal, without a correction for T1 discrimination, is to underestimate metabolites with a long T1 (sugar phosphates) and overestimate those metabolites with a short T1 (ATP). Individual metabolite T1 discrimination factors are calculated from integrated areas of spectra acquired using short and long repetition times as well as from metabolite T1 values. They are then employed, for the first time, for the correction of 31P-NMR spectra of bovine and rabbit lenses. Corrected spectra provide relative metabolite levels for lenticular ATP which are in excellent agreement with values determined by chemical and enzymatic assays.

Measurement of longitudinal relaxation times using surface coils

Abstract The poor B 1 field homogeneity associated with surface coils reduces the effectiveness of inversion-recovery techniques for determination of T 1 relaxation times. This paper presents a variation of the saturation-recovery T 1 experiment which introduces periodic phase shifts in the saturating irradiation to achieve rapid and effective saturation of the sample magnetization, thereby avoiding the complications of B 1 field inhomogeneity. Comparison of the presented technique with the inversion-recovery technique utilizing a composite inverting pulse and alternating phase acquisition is provided. A discussion of the relative merits of each technique is presented.

Oxidative-stress induced protein glutathione mixed-disulfide formation in the ocular lens

The biochemistry of protein-glutathione mixed disulfide formation in the ocular lens was examined by 13C-NMR spectroscopic measurements of glutathione oxidative metabolism in intact rabbit lenses maintained in organ culture. Lenticular amino acid uptake and glutathione biosynthetic mechanisms were employed to facilitate the incorporation of L-[3-13C]cysteine from the incubation medium into the cysteinyl residue of glutathione. Subsequent exposure to increasing levels of oxidative stress induced by tert-butylhydroperoxide resulted in decreased levels of ([3-13C]cysteinyl)-glutathione and a loss of 13C NMR resonance intensity, a reflection of protein-glutathione mixed disulfide formation. The rate of ([3-13C]cysteinyl)-glutathione loss depended on the concentration of tert-butylhydroperoxide; 13C-labeled oxidized glutathione was observed only at the highest concentration (2 mM) of oxidant tested. Removal of the oxidative stress led to a partial recovery of ([3-13C]cysteinyl)-glutathione levels and 13C resonance intensity. Recovery was significantly enhanced by the addition of 2-mercaptoethanol. The mechanism of protein-glutathione adduct formation was further characterized by the in vitro monitoring of the reaction of oxidized glutathione with bovine lens gamma-II crystallin protein using proton NMR spectroscopy. These experiments provided insight into the role of the cellular glutathione redox-couple, [GSH]/[GSSG], in maintaining reduced protein thiol groups, and suggested that protein-glutathione adduct formation may function as a mechanism for modulating the glutathione redox buffer under conditions of oxidative stress in ocular tissue. In addition, the results demonstrate the feasibility of direct chemical reduction of protein-glutathione disulfide bonds in vivo which may reflect a mechanism for the inhibition of disulfide-linked light scattering protein aggregate formation.

The effect of prolonged elevated glucose levels on the phosphate metabolism of the rabbit lens in perfused organ culture

13C and 31P nuclear magnetic resonance (NMR) techniques were used to monitor phosphate metabolite longitudinal (T1) relaxation times and metabolism, the sorbitol pathway, and related bioenergetic processes in cultured rabbit lenses through 9 days of incubation with constant perfusion. Lenses were incubated in a modified TC-199 medium containing either 5.5- or 35.5 mM [1-13C]-enriched glucose. The NMR studies were augmented by biochemical and cation analyses, and by the visual assessment of lens clarity. In the hyperglycemic rabbit lens, relative to normal values, longitudinal (T1) relaxation times for phosphorus metabolites increased from 33- to 50% [with the exception of inorganic phosphate (Pi)]. This provides the first documentation that a pathophysiological insult to the lens can alter phosphorus metabolite T1 values. The determination of steady state levels for the NMR visible phosphorus metabolites present in the lens was obtained after correction for T1 differential saturation effects, and normalization to reflect the content of phosphorus equivalents in each metabolite. Relative to control lenses (i.e. incubated in 5.5 mM glucose-containing medium) the NMR visible phosphate metabolite pool of rabbit lenses subjected to a hyperglycemic stress for an extended period of time (greater than 72 hr) is characterized by the following statistically significant differences: a 23% decrease in the mean level of ATP; a 51% increase in the mean level of alpha-glycerolphosphate (alpha GP); a 56% decrease in the mean level of Pi; the appearance of an unidentified resonance at 6.2 ppm after 115- to 120-hr incubation; and lenticular acidification of 0.10 to 0.17 pH units. No statistically significant differences in the mean levels of ADP, dinucleotides (predominantly NAD+-NADH), and phosphomonoesters (other than alpha GP) were evident. Parallel 13C NMR measurements provided a real-time confirmation of sorbitol production and accumulation in rabbit lenses incubated in 35.5 mM glucose-containing medium. In agreement with classical biochemical analysis (Chylack and Kinoshita, 1969) sorbitol production attained a plateau level after ca. 3 days of incubation. Cation determinations performed at the conclusion of the 9-day incubation indicated that the lenses incubated under the two conditions have similar Na+ and K+ levels. Rabbit lenses incubated in normal glucose medium remained clear for at least 8 days. By contrast, for the rabbit lenses incubated in elevated glucose medium, equatorial opacification became evident by day 5; by day 8 extensive bleb formation and opacification was evident.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo observation of lactate methyl proton magnetization transfer in rat C6 glioma

Magnetic resonance spectroscopy (MRS) measurements of the lactate methyl proton in rat brain C6 glioma tissue acquired in the presence of an off‐resonance irradiation field, analyzed using coupled Bloch equation formalism assuming two spin pools, demonstrated the occurrence of magnetization transfer. Quantitative analysis revealed that a very small fraction of lactate (f = 0.0012) is rotationally immobilized despite a large magnetization transfer effect. Off‐resonance rotating frame spin‐lattice relaxation studies demonstrated that deuterated lactate binds to bovine serum albumin and the proteins present in human plasma, thereby providing a possible physical basis for the observed magnetization transfer effect. These results demonstrate that partial or complete saturation of the motionally restricted lactate pool (as well as other metabolites) by the application of an off‐resonance irradiation field, such as that used for water presaturation, can lead to a substantial decrease in resonance intensity by way of magnetization transfer effects, resulting in quantitation errors. Magn Reson Med 41:676–685, 1999.