Nuclear magnetic resonance

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(see also Chapter 2) General description

Analogous to EPR spectroscopy, NMR spec-troscopy is based on the resonance absorption of electromagnetic radiation by the system during the transition between two discrete energy states. The energy differences studied in NMR spectroscopy are due to the interaction of nuclear magnetic moments with the magnetic field (Zeeman splitting for nuclei). The energy differences are smaller than those in EPR because of the smaller magnetic moment of nuclei. This explains why electromagnetic radiation in the radiofrequency range is required to excite the transitions that produce the NMR signal, whereas that in the microwave range is used in EPR spectroscopy.

Because the energy differences in NMR are small, the differences in number of nuclei at different energy levels are also small. As a consequence, the signal strength is weak, which makes NMR an inherently insensitive technique. Only those nuclei that have a non-zero spin quantum number resulting in non-zero magnetic moment can be used. The splitting between energy levels depends on the strength of the magnetic field and the mag-netogyric ratio of the nucleus. The highest magnetogyric ratio and the almost 100% natural abundance make proton (1H) NMR the most sensitive. The reasonably high magnetogyric ratio and 100% natural abundance of 31P nuclei give moderately good receptivity for in vivo phosphorus NMR. In contrast, the 13C nucleus has very low receptivity because of its low natural abundance, but, as a label, this isotope could be useful (Schneider, 1997; Roberts, 2000).

NMR signals can be characterized by intensity, frequency, line shape and relaxation times. All these characteristics are affected by the physical and chemical environment of the magnetic nucleus and can be used to obtain information of biological interest such as the state of water, intracellular pH and membrane dynamics. Signal intensity is related to the number of molecules that produce the signal. In relaxation experiments, the intensity depends on the time of signal registration and on the rate of magnetization decay. For quantitative estimation of peaks in NMR spectra, integration of the lines should be used because of the different relaxation times of the signals. The limits of integration are determined by the signal-to-noise ratio of the signal and the overlapping with other signals in a spectrum.

Local fields originating from the local electron density modify the external field imposed on magnetic nuclei. As a result, the resonance frequency of a nucleus depends on its chemical environment, which is called chemical shift. Magnetization relaxes exponentially, and the faster the decay, the broader the line in the spectrum. Broad lines have lower amplitudes and overlap with other lines, which leads to poorly resolved spectra. In living systems, the variations of magnetic susceptibility across the sample cause line broadening, which makes it difficult to record high-resolution spectra from dense heterogeneous tissue, such as seeds, and from tissues containing airspaces (leaves and roots).

The T1 (spin-lattice or longitudinal) and T2 (spin-spin or transverse) relaxation times characterize the magnetization decay because of the interaction of the nuclear magnetic moments with the environment (Tj) and with each other (T2). Relaxation times are mostly determined by the motional properties of the nucleus. Measurements of relaxation are particularly important in NMR studies of tissue water when information about the existence of different water fractions in the tissue is required. In practice, the measurements are easier to conduct than to interpret (Ratcliffe, 1994).

In basic NMR experiments, the sample is placed in a magnetic field, and the NMR signal is generated by irradiation of the sample with a radio-frequency field, given as pulses of different sequences. A single pulse creates a net magnetization, which is registered. The magnetization decays to zero, and the time-dependence of the decay (free induction decay) is recorded. In low-field studies, this decay is analysed directly. In high-field NMR, the decay is converted into spectra by Fourier transformation. The NMR spectrum is the plot of intensity against frequency of the radio-frequency field. All NMR applications developed for studying living systems can be divided into four groups: (i) detection of water signal; (ii) NMR imaging; (iii) high-resolution multinu-clear NMR spectroscopy; and (iv) solid-state NMR spectroscopy (Ratcliffe, 1994).

To exploit the advantage of a non-invasive technique, NMR experiments need to minimize the physiological perturbation and maintain the tissue in a physiologically controllable state. In this respect, the whole plant, cell suspensions and intact seeds are the easiest tissues, and excised tissues the most demanding (Ratcliffe, 1994). Often, it is necessary to submerge a sample in water to avoid differences in magnetic susceptibility between air and cellular material, a practice that is incompatible with drying organisms. Proper O2 supply and illumination have to be maintained, especially in densely packed samples. In solid-state NMR, when magic angle spinning is applied, it is impossible to control the physiological state because of the extreme conditions (more than 1000 rotations per minute) imposed on the sample. The NMR study of water in living systems general remarks. The study of water in anhydrobiotes is of particular interest because with drying and rehydration both water content and water properties change. NMR is a powerful tool to study water in vivo. There is no problem with the sensitivity of detecting the water signal in biologi cal systems because of the generally high water content, the high natural abundance and the high magnetogyric ratio of 1H. This allows the use of low-field NMR instead of expensive high-field NMR magnets.

measurements of water content. 1H low-field NMR allows the non-destructive measurement of the water content in biological systems with high precision. There are two types of analytical NMR commonly used in this respect - continuous wave (CW) NMR (wide-line) (Pohle and Gregory, 1968) and pulsed NMR (Martin et al., 1980), the latter now being generally adopted. In CW-NMR the amount of liquid water is estimated from the area under the absorption peak. The signal from water strongly 'bound' to biopolymers is not visible because of broadening. The signals from liquid water and oil are not resolved, but the contribution of oil to the signal can be estimated by drying.

Pulsed NMR can be used to analyse the different water fractions. In pulsed NMR all protons are excited by a short intense radio frequency (RF) pulse resulting in a free induction signal, which decays when the pulse is switched off. The initial amplitude of free induction decay (FID) is proportional to the total number of protons in a sample. The signals due to nuclei in different physical states decay at different rates: signals due to protons in solid state decay faster (microseconds) than those in liquid phase (from milliseconds to seconds). This signal decay can be analysed to reveal the contribution of different proton fractions. To avoid the influence of inhomogeneity of magnetic field and water diffusion on the rate of decay, special sequences of pulses such as spinecho (SE) or Carr-Purcell-Meiboom-Gill (CPMG) are used (Farrar and Becker, 1971). In air-dry samples, the signal decay from water associated with polymers (mainly starch) can be distinguished easily from that of oil protons on the basis of the considerable differences in spin-spin relaxation time T2. Such an approach is widely used for rapid and non-destructive determination of moisture and oil content in air-dry seeds (e.g. Tiwari et al., 1974;

Gambhir and Agarwala, 1985; Brusewitz and Stone, 1987; Gambhir, 1992; Rubel, 1994; Warmsley, 1998). In hydrated seeds, drying or D2O exchange can be used to separate the NMR signal of free water from that of oil (Ratkovic et al., 1982a).

Because the different water fractions have the same chemical shift, only pulsed NMR can be used to characterize them in living tissues. The changes in water fractions with different relaxation characteristics can be followed during the dehydration or rehydration of anhydrobi-otic systems. This gives insight into the role of the different water fractions in biological systems (Seewaldt et al., 1981; Ratkovic et al., 1982a; Aksyonov and Golovina, 1986a,b; Ishida et al., 1987, 1988b; Bacic et al., 1992; Golovina and Aksyonov, 1993; Marconi et al., 1993). However, data on different water fractions must be interpreted with extreme caution (Ratcliffe, 1994). Different water fractions with specific relaxation times can be discriminated only if there is no fast exchange of protons between the fractions in the NMR time window. In the case of fast exchange between protons, only one relaxation time is observed. The number of protons of different mobility and their relaxation times will determine the observed effective relaxation time. When associated with macromolecules, water protons have shorter relaxation times, which will influence the overall relaxation time. This is the reason why T1 (spin-lattice relaxation time) and T2 (spin-spin relaxation time) values are lower in cellular water than in bulk water and decrease further with water loss. Thus, T2 values can also be used to measure moisture content (MC) (Ratkovic, 1987). Below 0.2 g H2O g^1 dry weight, the relationship between relaxation times (T1 and T2) and moisture content is reversed (Clegg et al., 1982; Ratkovic et al., 1982b; Wolk et al., 1989). Because the increase in T1 and T2 at low water contents has also been observed in starch/water systems besides anhydrobiotic organisms, the increase might be attributed to water molecules jumping from one sorption site to another.

Compartmentation is the reason why more than one fraction of water is generally observed in hydrated living systems. A theory of transverse relaxation in compart-mented systems has been developed, based on the chemical exchange and diffusion properties of the water (Belton and Ratcliffe, 1985). Two to three water fractions have been shown in hydrated tissue originating from different plant cell compartments (Bacic and Ratkovic, 1984; Belton and Ratcliffe, 1985; Snaar and van As, 1992). However, it appears that there is no simple relationship between the multi-exponential character of T2 and the com-partmentation of the water (Ratcliffe, 1994). The heterogeneity in cellular size and composition, subcellular compartmentation, and plasmalemma and tonoplast permeability could have influenced the multi-exponential decay curves (Snaar and van As, 1992). The detection of the simultaneous presence of water of different relaxation behaviour in anhydrobiotes with reduced MC may have been caused by the inhomo-geneous water distribution within the organisms. Thus, the water with long T2 (or slow-relaxing water) observed in wheat kernels during the first hours of imbibition is thought to be localized around the embryo and in the vascular bundle, whereas the fast-relaxing water is thought to be associated with starchy endosperm (Golovina and Aksyonov, 1993).

water self-diffusion coefficient. The behaviour of water in living systems can also be characterized by the water self-diffusion coefficient. The diffusion coefficient is measured by the pulsed (spin-echo) NMR technique in the presence of a (pulsed) field gradient (Fukushima and Roeder, 1981). In addition to nuclear magnetic relaxation, the spin-echo amplitude decreases in the presence of a field gradient if water changes its position during the measurement. Diffusion coefficients as a measure of water mobility can be calculated from the signal decay in the presence of a field gradient. As in the case of relaxation times, self-diffusion coefficients of cellular water are lower than those of bulk water and decrease with drying (Clegg et al., 1982). This can be caused by the presence of diffusion barriers (membranes or cell walls) or macromolecules. These macromolecules can cause either obstruction of the diffusion or water binding (Seitz et al., 1981; Back et al., 1991). As a result, the diffusion coefficient in hydrated anhy-drobiotes has been shown to be 2-5 times smaller than that of bulk water (Clegg et al., 1982; Fleischer and Werner, 1992). In Artemia cysts the diffusion coefficient has been measured from 0.02 to 1.49 g water g_1 dry weight, the minimum value being almost 50 times lower in the dry cysts than in the hydrated cysts (Seitz et al., 1981).

membrane permeability. Paramagnetic ions (Mn2+) cause a decrease in relaxation times due to their interaction with nuclei. Conlon and Outhred (1972) proposed a method of measuring membrane permeability to water, based on the change in relaxation time of intracellular water that is in diffusional exchange with an extracellular MnCl2 solution. From the estimated water-exchange time and the cell dimension, the diffusion permeability coefficient Pd can be calculated (Stout et al., 1977, d978; Bacic and Ratkovic, 1984). Unfortunately, this approach cannot be applied to the systems that are subjected to drying, because the tissue has to be in Mn2+ solution.

The pulsed-gradient spin-echo method proposed by Stejskal and Tanner (1965) can be used to study the in situ membrane permeability for water during drying. The method allows the water diffusion to be measured over the time between two pulses of field gradient. The presence of partly permeable barriers causes the decrease in the apparent diffusion coefficient for water, so that the permeability of membranes for water and the size of water compartments can be calculated (Tanner, 1978; von Meerwall and Ferguson, 1981). This approach has been applied to follow the changes in membrane properties in developing barley seeds (Ishida et al., 1995) and to calculate the size of oil bodies in rape seeds (Fleischer et al., 1990; Fleischer and Werner, 1992). NMR imaging general remarks. NMRI is mainly based on the detection of the water signal. 1H resonance frequency is independent of the location of the water in a tissue, so that tissue water signal is averaged across the whole sample. The spatial distribution of the water signal can be obtained if a magnetic field gradient is applied, which arises from the dependence of the resonance frequency of NMR signals on the magnetic field strength. In spite of the simplicity of the principle of NMRI, its practical application is rather complicated. Information on the spatial distribution of water or water properties (relaxation times or diffusion coefficients) can be obtained. Dynamic information can be obtained from time-dependent properties of the image. There are two different experimental approaches in NMRI: imaging large objects (roots, stems or whole plants) with low spatial resolution, and imaging small samples (seeds, excised tissues) with high spatial resolution (NMR microscopy) (Ratcliffe, 1994; Ishida et al., 2000).

Spatial resolution is mainly determined by the signal/noise ratio, but other factors such as short relaxation times and the presence of air space cause intensity loss and a decrease in spatial resolution. The development of NMRI has led to a resolution that approaches the dimension of single cells in plant tissues (Connelly et al., 1987). The theoretical limit is considered as 10 X 10 X 10 pm (Ratcliffe, 1994). While NMR is not yet able to compete with optical microscopy in its resolution of cellular structures, it has the great advantage of being non-invasive and, thus, can be used to monitor functioning plant tissue. The ability to resolve structures depends not only on resolution but also on the image contrast, which is determined by the differences in signal intensity between different regions of the sample. Knowledge of relaxation properties of the tissue water is central to the understanding of image contrast. Nitroxide radicals (Magin et al., 1986; Swartz et al., 1986) and paramagnetic ions (Ishida et al., 2000) can be used as contrasting agents.

water distribution in seeds during maturation and germination. It is possible to map stationary, diffusing and flowing water in plant tissue (Ratcliffe, 1994). NMRI enables the water distribution inside seeds to be determined. The brightness of the image is proportional to the proton density. Experiments with seeds of different species have shown that the signal/noise ratio in the image is sometimes limited by the short relaxation time for tissue water (T2 < 10 ms) (Connelly et al., 1987). The sensitivity problem can be overcome to some extent by signal averaging, since the time scale for detectable structural changes in germinating seeds is long in comparison with the time required to obtain an image. In NMR images of seeds, a clear distinction between axis and storage tissue can be obtained (Connelly et al., 1987; Kano et al., 1990; Hou et al., 1996; Fountain et al., 1998; Carrier et al., 1999). The changes in water distribution during drying and rehy-dration have shown the transfer routes for water (Ruan and Litchfield, 1992; Ruan et al., 1992; Song et al., 1992; Kovacs and Nemenyi, 1999).

The water content may be more uniformly distributed in seeds than proton NMRI indicates. This discrepancy arises from the inhomogeneity of the susceptibility of the sample associated with the presence of cell walls and storage substances (Back et al., 1991). Eccles et al. (1988) applied pulsed gradient spin-echo and steady gradient NMRI to maturing wheat kernels and found the spatial distribution of the self-diffusion coefficient of water. The diffusion was slowest in endosperm and highest in the vascular bundle. Back et al. (1991) used the dependence between the self-diffusion coefficient of water and the relative water content obtained by Callaghan et al. (1979) to correct the proton map for wheat grain and showed the more uniform distribution of water in the corrected image.

For experiments in which germination of seeds has to be followed over many hours in the magnet, it is necessary to maintain a continuous water supply to the seeds, while at the same time minimizing the spectroscopic signal of the externally supplied water (Connelly et al., 1987). The changes in relaxation times of tissue water during seed maturation or germination cause changes in the image contrast. Relaxation times of water depend on the interaction of water with macromolecules. The synthesis of storage substances during maturation and their hydrolysis during germination result in an apparent decrease or increase in brightness of the NMR image (Ishida et al., 1990, 1995; McIntyre et al., 1995), so that solubilized parts of the storage tissue can become visible. The changes in image contrast during precocious germination of Phaseolus vulgaris seeds after ethylene treatment have been attributed to changes in the water status and water redistribution from the cotyledon to the axis (Fountain et al., 1998).

the distribution of oil and sucrose in seeds.

The spatial image of other compounds, mainly lipids and carbohydrates that accumulate in storage tissue, can be mapped in vivo using the chemical-shift imaging (CSI) technique (Bottomley et al., 1984). The 1H NMR spectra of water, oil and sugars have different chemical shifts, but the peaks are not resolved unless the water peak is suppressed. The CSI technique applied to 1 day germinating mung bean seeds has shown uniformly distributed oil, which allowed the changes in the image with germination to be attributed to the bulk water fraction (Connelly et al., 1987). Oil and sucrose have been mapped in fresh maize kernels (Koizumi et al., 1995), germinating barley seeds (Ishida et al., 1990) and in developing pea seeds (Tse et al., 1996). High-resolution multinuclear NMR spectroscopy general remarks. High-resolution multi-nuclear NMR is used to detect ions and metabolites of low molecular weight, the intracellular pH, the subcellular compart-mentation of compounds and the flux through metabolic pathways (Ratcliffe, 1994; Schneider, 1997; Roberts, 2000). Low concentration of the molecules of interest and low receptivity of nuclei other than 1H make this approach rather insensitive. The sensitivity increases with increasing field strength. High-resolution NMR spectrometers are usually equipped with high-field superconducting magnets in the range 4.7-14.1 T, corresponding to a 1H frequency of 200-600 MHz. The sensitivity can be increased by multiple scanning and usually permits the detection of millimolar concentrations of metabolites (Ratcliffe, 1994). To increase the sensitivity further, the tissue volume within the detector has to be maximized. Cell suspensions and excised tissues are more suitable for such experiments than whole plants or seeds.

1h nmr. Different nuclei can be used for different purposes. The high sensitivity makes 1H attractive for metabolite detection. Nevertheless, the need to suppress the water signal and the complexity of spectra limit the possibilities for in vivo 1H NMR. The small differences in chemical shift and considerable overlapping of broad signals in tissues make 1H spectra poorly resolved. For example, in germinating seeds only peaks from sugars and oil under conditions of partial water signal suppression can be resolved (Koizumi et al., 1995; Ishida et al., 1996). 1H NMR spectra of oil in dry seeds can be obtained because the signals from other nuclei are broadened due to immobilization. However, the resolution of lines is not good because of differences in magnetic susceptibility. The magic-angle sample spinning (MASS) technique eliminates line broadening arising from differences in magnetic susceptibility due to fast mechanical rotation about an axis, making a magic angle (54°55'), and resulting in 1H spectra from dry seeds with a good resolution (Rutar, 1989). 1H NMR is widely used to analyse tissue extracts for the presence of specific compounds such as, for example, betaine in wild-type and transformed Arabidopsis thaliana seeds (Alia et al., 1998).

13c nmr. 13C NMR is more attractive for application in vivo for two reasons. First, the chemical shift scale of the 13C nucleus is more than an order of magnitude greater than that of the 1H nucleus, which reduces overlapping in the spectra. Secondly, the low natural abundance of 13C opens possibilities for labelling the tissue and monitoring metabolic pathways. The biological use of NMR to study metabolism is described in Section 4.3.2. 13C NMR has also been used to establish changes in soybean seeds during maturation and germination. The moisture content-dependent disappearance or appearance of narrow peaks associated with sugars in in vivo NMR spectra is indicative of the presence of free water in these seeds (Ishida et al., 1987, 1988a). The sensitivity of natural abundance 13C NMR can be enhanced, by applying low-speed magicangle spinning (Ni and Eads, 1992) or by the detection of 13C by protons coupled to the 13C nucleus (Heidenreich et al., 1998). 13C labelling gives opportunities for probing different metabolic pathways, such as lipid synthesis in soybean ovules (Schaeffer et al., 1975) and the metabolism of dormancy-breaking chemicals in red rice (Footitt et al., 1995).

31p nmr. In vivo 31P NMR has many applications because of the convenient magnetic properties of the 31P nucleus and the physiological importance of the information that can be deduced from the spectra. The measurement of cytoplasmic and vacuolar pH is one of the most important applications of in vivo 31P NMR, which is based on the dependence on pH of the chemical shift of Pi. This, together with the slow exchange of Pi across the tonoplast, allows the origin of the Pi signal - either cytoplasmic or vacuolar -to be determined and, consequently, the cytoplasmic and vacuolar pH. A number of important phosphorylated metabolites can be resolved in 31P spectra. For some of them (Pi, polyphosphates), information on the subcellular distribution can also be obtained because of the pH-dependent chemical shift. 31P NMR has been applied to study the pH of intracellular compartments in germinating seeds of Phacelia tanacetifolia (Espen et al., 1995). Changes in chemical shifts of the pH-dependent 31P signal from cytoplasmic and vacuolar inorganic phosphate correlate with seed germination. 31P can also be used to monitor phosphorus compounds and their changes during maturation and germination of seeds, both in extracts and in vivo. Because of line broadening in in vivo experiments, a lower number of phosphorus compounds can be resolved (Ishida et al., 1987, 1988a) in comparison with extracts (Ricardo and Santos, 1990). 31P spectra can be used for the identification of the appearance or disappearance of vacuoles in seeds during germination and maturation (Ishida et al., 1990). Structure and dynamics of cellular membranes general remarks. NMR provides a rapid, non-invasive method for investigating the state of membranes in isolated cellular fractions and in living tissues. The approach in the study of membrane structure and dynamics is solid-state NMR, because of the anisotropic nature of the membranes. The main nuclei used for this study are 31P and 2H. Sometimes, labelling with 13C has been used, although the line shape is difficult to analyse.

31p nmr The chemical shift of the phospho-lipids depends on the orientation of the phosphate groups with respect to the magnetic field. In the case of unrestricted motion, all directions are averaged and the spectrum is isotropic and contains the narrow symmetrical 31P NMR line (Cullis and de Kruijff, 1979). In some cases, peaks from different phospholipids can be resolved (Smith, 1985). In the case of restricted mobility of phospholipids in membranes, the spectrum is anisotropic. The shape of the anisotropic 31P NMR spectrum depends on the type and rate of motion of the phos-pholipids. Thus, 31P NMR spectra are sensitive to the physical state of the phospholipids. From the spectra, the order parameter can be calculated (Smith, 1985). There are a few examples of the successful application of 31P NMR in the field of desiccation tolerance. Lee et al. (1986, 1989) studied the interaction of trehalose with the phospholipid, dipalmitoylphosphatidyl-

choline (DPPC). It was shown that the head groups are in a rigid state above and below the phase transition for both dry DPPC and a mixture of dry DPPC and trehalose. Tsvetkova et al. (1998) used 31P NMR in a comparative study of the interaction of glucose, trehalose and hydroxyethyl starch with dry DPPC. The differential effect of carbohydrates on the behaviour of head groups has been related to the role of trehalose in membrane protection upon drying.

Phospholipids arranged in bilayers or in an inverted hexagonal phase have different line shapes (31P pattern) (Cullis and de Kruijff, 1979). These differences between bilayer and hexagonal phase spectra arise from the fact that the lipids are restricted in motion to the plane of the membrane in the lamellar state. In the case of the hexagonal phase, a rapid motion about the cylinder axis averages the chemical shift anisotropy. These differences in 31P pattern can be used to detect the presence of either phase.

For many years researchers have been interested in the membrane transition upon drying from the bilayer into the hexagonal phase (Simon, 1974). In an attempt to detect this membrane transition, Priestley and de Kruijff (1982) applied 31P NMR to several dry biological systems. The in vivo spectra were complicated by the superposition of the signals from phospholipids and phosphorus-containing compounds. Pollen of Typha latifolia was the most suitable for spectra analysis. At 5.2% MC, the line shape of the spectrum was broad and not suitable for analysis. At MC ^ 8.8%, only isotropic signals from phosphorus low-weight molecules could be identified, but, at 10.9% MC, a clear peak from phospho-lipids organized in bilayers became evident. Thus, no evidence was obtained for the presence of a hexagonal phase in the pollen on drying to 10.9% MC.

2h nmr. The relatively small quadrupole moment of deuterium makes it an ideal probe of membrane lipids (Smith, 1985). Fatty acids labelled with 2H at different positions must be synthesized. The 2H NMR spectrum of membranes contains three clearly separated lines ('rabbit ears'), and the separation relates to the ordering of the 2H-labelled segment. Quadrupole splitting, overall pattern and relaxation times are usually used to characterize 2H spectra. Spin-lattice relaxation is sensitive to relatively rapid motions, whereas spin-spin relaxation is sensitive to slow motions (Smith, 1985). This technique can be used to study membrane phase transitions, the influence of acyl chain saturation on membrane fluidity and changes in membrane fluidity.

2H NMR was applied by Lee et al. (1986, 1989) in a study on the effect of interaction of trehalose with dry DPPC on the behaviour of acyl chains. 2H quadrupole spectra of dry DPPC labelled at the 7th position showed that the disorder of lipid acyl chains is much greater in the case of interaction of DPPC with trehalose above the phase transition than in hydrated or dry DPPC without trehalose. The new type of liquid-crystalline phase observed in the dry mixture of trehalose and DPPC is believed to play a main role in maintaining membrane stability in dehydrating organisms.

13c nmr. 13C-labelled phospholipids can be used to study the particular dynamics of membranes in the interfacial region. Lee et al. (1989) used 13C-labelled sn-2-carbonyl of DPPC to study the influence of the interaction of dry DPPC with trehalose on interfacial behaviour. No changes in 13C NMR powder spectra were observed during the phase transition of a dry mixture of DPPC/trehalose, whereas hydrated DPPC exhibited pronounced changes during the phase transition.

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