[Natural polymers according to NMR data: cross-relaxation in hydrated collagen macromolecules from two connective tissues]. / Prirodnye polimery po dannym IaMR: krossrelaksatsiia v makromolekulakh kollagena iz dvukh soedinitel'nykh tkanei.

Spin-lattice relaxation and cross-relaxation in oriented and randomly oriented collagen fibers from two connective tissues (15-month-old calf and 8-year-old steer) at a water content of 0.6 g H2O/g dry matter were studied. Collagens were chosen according to different numbers of covalent nonreducible cross-links, which increase during the life of the animal. The spin-lattice relaxation curves for all the collagens after a 180 degree-tau-90 degree pulse sequence were described by two exponential components. The dependences of two components of spin lattice relaxation time and their populations on the length of the 180 degree-pulse were obtained. On the basis of data of Goldman-Shen sequence and the two-phase model, the populations of proton fractions (p(w) and p(c)) as well as the rates of transfer of magnetization between water protons and collagen protons (k(w) and k(c)) were calculated. No significant difference between k(w) (k(c)) in oriented and randomly oriented fibers as well as in fibers with different cross-linking was found. The estimates of the cross-relaxation times for low cross-link collagen and high cross-link one were done. The correlation times of dipole-dipole interactions for both connective tissues were calculated using the cross-relaxation theory.

Water transfer analysis in pork meat supported by NMR imaging.

NMR proton density imaging was used to study isothermal and unidirectional drying of pork semi membranosus muscle samples at temperatures of 12, 16 and 20 °C. An independent calibration of the transversal relaxation time T(2) as a function of the moisture content was carried out to convert the signal amplitude into moisture content. Due to spatial heterogeneity in drying, 2D images were needed to assess the evolution of 1D moisture profiles. The relationship between the effective water diffusivity (D) was calculated in function of water content (X) using the Boltzman transformation which needs no a priori on the relationship D=f(X); the effect of lipid content, temperature and fibre direction on this relationship were also studied. In all cases a decrease in water content brought about a decrease in D. A slight increase in lipid content led to a dramatic decrease in D. The fibre direction relative to water movement had a negligible effect. No significative differences in D between the three temperatures were observed, due to variability in the chemical composition of the samples.

1H NMR studies: dynamics of water in gelatin.

Proton magnetic resonance was used to characterize the dynamics of water in gelatin. Both sol and gel states were investigated. Transverse relaxation rates (R2) were dependent on the proton frequency measurement. (R2) measured with the Carr-Purcell-Meiboom-Gill pulse sequence was dependent on pulse spacing. These observations were interpreted in terms of chemical exchanges between water protons and those of the macromolecules in the sol state, whereas in the gel state the contribution of diffusion through microheterogeneities in the sample seems to provide an additional transverse relaxation mechanism.

1H-nmr study of water dynamics in hydrated collagen: transverse relaxation-time and diffusion analysis.

Water proton transverse relaxation times (T2) and self-diffusion coefficients (D) were measured in randomly oriented hydrated collagen fibers. Three T2 relaxation times were discerned indicating the presence of at least three water fractions in the collagen sample. The D values associated with each water fraction were determined. The diffusion time dependence of D suggests water motion is restricted by macromolecular structure. The experimental results are discussed with reference to the structural properties of hydrated collagen fibers.

Effect of thermal denaturation on water-collagen interactions: NMR relaxation and differential scanning calorimetry analysis.

The dependence of the proton spin-lattice relaxation rate, and of the enthalpy and temperature of denaturation on water content, were studied by nmr and differential scanning calorimetry (DSC) in native and denatured collagen. Collagen was first heated at four different temperatures ranging from 40 to 70 degrees C. The percentage of denatured collagen induced by these preheating treatments was determined from DSC measurements. The DSC results are discussed in terms of heat-induced structural changes. A two-exponential behavior for the spin-lattice relaxation was observed with the appearance of denatured collagen. This was attributed to the presence of a noncollagen protein fraction. The variations in the different longitudinal relaxation rates as a function of the moisture content and of the denatured collagen percentage are described within the multiphase water proton exchange model. This study highlights the complementarity of the information obtained from the two analytical tools used.

Noninvasive skeletal muscle lactate detection between periods of intense exercise in humans.

We investigated whether localized 1H nuclear magnetic resonance spectroscopy (NMRS) using stimulated echoes (STEAM) with a long mixing time (t(m)) allowed the suppression of the fat signal and detection of lactate in skeletal muscle. The 1H NMRS sequence was first validated in three isolated and perfused rabbit biceps brachii muscles. Spectra were obtained on a wide-bore spectrometer using a dual-tuned probe (1H and 31P). Death was simulated by ceasing the muscle perfusion, which allowed post-mortem changes to be followed. During and after the simulated death, changes in levels of pH and in content of energy-rich compounds were observed with 31P NMRS. Our results showed an inverse linear relationship between pH and lactate in each of the three rabbits (r = 0.93, P < 0.001; r = 0.92, P < 0.01; r = 0.89, P < 0.01) and a decrease in phosphocreatine and concomitant increase in lactate. We then investigated whether this sequence allowed repeated detection of lactate in human soleus muscle during the recovery between periods of intense exercise (force-velocity test, F-v test). Seven subjects mean age 25.1 (SEM 0.8) years participated in this study. Soleus muscle lactate was detected at rest and for 3 min 30 s of the 5-min recovery between periods using a 2.35-T 40-cm bore magnet spectrometer. Arm venous plasma lactate concentration was measured at rest, during the F-v test when the subject stopped pedalling (S1), and at the end of each 5-min recovery between periods (S2). Results showed that the venous plasma lactate concentration at S1 and S2 increased significantly from the beginning of the F-v test to peak anaerobic power (W(an,peak)) (P < 0.001). The spectra showed that muscle lactate resonance intensity rose markedly when W(an,peak) was achieved. The muscle lactate resonance intensity plotted as a percentage of the resting value increased significantly at W(an,peak) compared with submaximal braking forces (P < 0.05). We concluded from these results that localized 1H NMRS using STEAM with a long t(m) allows suppression of the fat signal and repeated detection of lactate on isolated perfused skeletal muscle in animals and between periods of intense exercise in humans.

Optimization of Signal Intensity and T1-Dependent Contrast with Nonstandard Flip Angles in Spin-Echo and Inversion-Recovery MR Imaging

Theoretical expressions of flip angle values maximizing signal intensity and T1-dependent contrast are derived for spin-echo and inversion-recovery sequences. Experimental data and theoretical predictions are closely correlated for experiments carried out on phantoms, despite the nonideal shape of the RF refocusing pulse used for slice selection. The use of nonstandard angles is justified when rapid MR acquisitions are needed and/or large T1 species must be imaged with refocused sequences. Copyright 1998 Academic Press. Copyright 1998 Academic Press

pH is regulated differently by glucose in skeletal muscle from fed and starved rats: a study using 31P-NMR spectroscopy.

The aim of this study was to determine whether exogenous glucose metabolism influences the pH in superfused EDL muscle from growing rats fed or starved for 48 h (body weight 55 and 45 g, respectively). Energy state and intracellular pH of muscle were repeatedly monitored by 31P-nuclear magnetic resonance spectroscopy (31P-NMRS); glycogen and other energy metabolites were assayed enzymatically in muscle extracts at the end of the experiment. In EDL muscles from starved rats superfused with glucose for 4 h, intracellular pH was elevated (7-7.3), lactate concentration low, glycogen repletion very intense and citrate synthase activity high. We conclude that glucose was routed mainly toward both oxidative phosphorylation and glycogen synthesis in EDL muscles after food deprivation of rats. In contrast, the major pathway in muscles from fed rats may be glycolysis because the glycogen pool remained constant throughout the experiment. The additional and minor pH component (in the range of 6.5 to 6.8) seen in muscles from fed rats, even in the presence of exogenous glucose, might be due to impaired glucose utilization because this component appears also in muscles from starved rats superfused without glucose or with a nonmetabolizable analog of glucose. Consequently, direct pH measurement by 31P-NMR may be considered to be a precise criterion for evaluation of differences in metabolic potentialities of muscle studied ex vivo in relation to the nutritional state of rats.

Contribution of proteolysis and de novo synthesis to alanine production in diabetic rat skeletal muscle: a 15N/1H nuclear magnetic resonance study.

To assess the role of leucine as a precursor of alanine alpha-amino nitrogen in skeletal muscle during diabetes, extensor digitorum longus muscles from control (n = 7 experiments) and streptozotocin-diabetic rats (n = 8 experiments) were isolated and superfused with [15N]leucine (3 mmol/l) in the presence of glucose (10 mmol/l) for 2 h. Muscle perchloric acid extraction was performed at the end of superfusion in order to quantify newly synthesized alanine by 15N/1H nuclear magnetic resonance. Release of [15N]alanine in the superfusion medium was also measured. The pool of newly synthesized [15N]alanine was significantly increased (approximately 40%) in extensor digitorum longus muscles from streptozotocin-diabetic rats. Whereas a significant enhancement of total alanine release from muscle was induced by diabetes (20%), only a slight increase in [15N]alanine release was detectable under our experimental conditions. Consequently, we conclude that streptozotocin-diabetes in growing rats induces in skeletal muscle: 1) an increase in nitrogen exchange between leucine and alanine leading to newly synthesized [15N]alanine; and 2) an increase of total alanine release from muscle originating from both proteolysis and de novo synthesis.

Effect of brine injection on water dynamics in postmortem muscle: study of T2 and diffusion coefficients by MR microscopy.

The dynamics of water in postmortem muscle were studied by magnetic resonance microscopy (MRM). Rabbit muscles were arterially injected with 3 and 5 M NaCl brine. T2 and diffusion mapping were performed during the onset of rigor mortis. A wide spread of T2 values and widely differing postmortem evolutions were observed for injected muscles, whereas T2 was spatially homogeneous for intact muscle. Also, highly variable spatial distribution of diffusion coefficients along (Dz) and across (Dx) the muscle fibers was observed and diffusion anisotropy Dz/Dx) was less marked in injected muscles. The results indicate heterogeneity of brine distribution far from the injection site soon after injection. The postmortem evolution of the parameters is discussed in terms of structural changes induced by brine injection. MRM provides insight into how water dynamics respond to different NaCl concentrations inside muscle.

Establishment of hydrogen-utilizing bacteria in the rumen of the newborn lamb.

The development of hydrogenotrophic bacteria in the rumen of lambs was investigated by culture and labeling experiments. 14CO2 and 13CO2 incorporation by the rumen microflora of a 24-h-old lamb showed that while there was no labeled methane, double-labeled acetate was formed indicating the presence of hydrogen-dependent acetogenesis. In vitro counts from rumen fluid of 20-h-old lambs confirmed an extensive colonization of acetogenic bacteria while methanogens were absent. Methanogens appeared in the rumen of 30-h-old lambs, and as they developed there was a proportional decrease in the numbers of acetogens, indicating a competition for hydrogen between these two groups. Hydrogen-utilizing sulfate-reducing bacteria, which were established by the 3rd day after birth, did not seem to be affected by this competition.

23Na magnetic resonance imaging: distribution of brine in muscle.

Magnetic resonance imaging (MRI) has been used to characterize tissues in morphological studies. Here, 23Na NMR imaging was used to study the distribution in muscle of brine (5 M NaCl), injected before onset of rigor mortis. First, the distribution of brine was monitored in excised muscle from rabbits for 6 hr following arterial brine injection. Immediately after injection, distribution was clearly heterogeneous. After 6 hr, a steady state was achieved but the observed brine distribution was not fully homogeneous. Second, the same was done for ham processed in various ways (tumbling, cooking). Tumbling increased the homogeneity of distribution in our experimental conditions but only cooking afforded full homogenization of brine in ham. Concentrations of NMR-visible sodium in the muscles were appreciably lower than the expected values calculated from the volume of injected brine. The invisible sodium presumably has T2 values which are too short compared with the echo time in our conventional spectrometer.

Use of superfused rat skeletal muscle for metabolic studies: assessment of pH by 31P n.m.r.

We developed a muscle superfusion system suitable for metabolic studies of small isolated rat muscle ex vivo in real time and in a non-destructive manner by n.m.r. spectroscopy. In order to determine biochemical stability of superfused extensor digitorum longus (EDL) muscle (from fasted 45 and 100 g rats), the energy state and the pH of muscle were continuously monitored by 31P n.m.r. spectroscopy. ATP and phosphocreatine remained stable during 2 h whatever the muscle size (20 or 45 mg). Neither metabolite was a sensitive probe of possible metabolic compartmentation within muscle under our experimental conditions. By contrast, the chemical shift of Pi by its sensitivity to pH was a discriminant factor in the assessment of muscle stability. Indeed, heterogeneity of pH was observed only in the 45 mg EDL muscle resulting from a core region with loss of glycogen. Together, these observations suggest deviations of energy metabolism to supply ATP. Consequently, pH may be considered as a new real-time criterion for monitoring a metabolic heterogeneity due to changes in energy metabolism of muscle preparations ex vivo.

Use of perfused isolated muscle, as studied by (31)P NMR, to investigate metabolism and post-mortem changes.

An experimental system was designed to study as independently as possible the effects of various in-vivo or post-mortem factors susceptible to influence muscle metabolism. This system was made up of an NMR probe, a physiological stimulator, a perfusion system and a force monitoring device. Rabbit muscles were isolated and perfused with bovine red cells, then put into the NMR probe to follow the evolution of pH and phosphorylated compounds. It was possible to keep muscle metabolism stable for 2 h. Death was simulated by stopping the perfusion which allowed post-mortem changes to be followed. The effects of adrenaline perfusion or of a 5 s tetanus on some traits of metabolism and on changes following muscle death were studied. Tetanus immediately before perfusion was stopped accelerated changes in pH and in phosphocreatine and ATP contents; adrenaline perfusion during 30 min before perfusion was stopped had little effect on these traits.