L. Foucat

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.

NMR and DSC studies during thermal denaturation of collagen

Abstract Epimysial and intramuscular connective tissues from calf and cow muscle were studied by NMR and DSC. Water proton NMR transverse relaxation times ( T 2 ) were measured at 10°C for both native and thermally-denatured at 90°C for 30–360 min. DSC measurements were used to determine the temperature and the variation enthalpy of sol→gel transition. According to the heating time, significant differences were observed between tissues. NMR discriminated the type of collagen whereas DSC distinguished the age of tissue. Differences were related to the degree of protein hydration, emphasising the complementary information from these two analytical tools.

Magnetic resonance imaging studies of water interactions in meat

The use of MRI shows spatial resolution of water content, and NMR parameters including relaxation times (T1 and T2), and diffusion coefficients (D) define the state of water interactions with other molecules. These parameters are potentially sensitive to local variations of water mobility and result from modification of water–macromolecule interactions and changes in tissue structure. MRI gives a unique opportunity to better-understand the dynamic phenomena that occur during processing and storage of food. By measuring apparent diffusion coefficient, both axially and radially in meat, it is possible to probe the influence of intracellular diffusional barriers or post-mortem structural changes. The effects of different freezing methods on trout muscle were investigated using MRI. The variations of the relaxation time, T2, and the radial diffusion coefficient, characterize the structural changes of tissue produced by the freezing process.

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.

Despite similar rates of alanine release, fasting and diabetes affect de novo alanine synthesis differently

Dear Sir, Diabetes and fasting have long been known to induce a substantial release of alanine from muscle whereas the intramuscular alanine pool remains unchanged [1, 2]. The substantial alterations of alanine metabolism in fasting and diabetes are not completely understood, especially with regard to the contribution of leucine as nitrogen donor in alanine de novo synthesis. Alanine remains an important gluconeogenic precursor in humans [3, 4], although glutamine was recently reported as a major source of carbon for gluconeogenesis in diabetic or postabsorptive humans [5, 6]. For this reason, we recently assessed, by using [15N]leucine, the capacity of skeletal muscle to synthesise alanine de novo through leucine transamination in experimental diabetic rats. Indeed, few studies have addressed either the direct effect of leucine supply on both synthesis (within the muscle) and release of alanine (from the muscle) or the contribution of proteolysis and de novo synthesis to alanine production in muscle. For this purpose, we used 15N/1H NMR combined with GC-MS. We have previously demonstrated that streptozotocin-induced diabetes in growing rats is associated with: 1) an increase in nitrogen exchange between leucine and alanine leading to newly synthesised [15N]alanine; and 2) an increase in total alanine release from muscle originating from both proteolysis and de novo synthesis [7]. In the present letter, we would like to report some data obtained with extensor digitorum longus muscles from fasted rats in order to compare nitrogen exchange between leucine and alanine in 48 h-fasting and in experimental diabetes. We observed that the size of [15N]alanine pool within the muscle was smaller in fasted than in diabetic and control extensor digitorum longus muscles (Table 1). Yet, in order to evaluate the true nitrogen transfer between leucine and alanine, both the labelled intramuscular alanine pool (ML) and the labelled alanine released (RL) at the end of experiment should be taken into account. For this reason, we calculated the sum of these two components under each experimental condition (see Table 1). Surprisingly, nitrogen exchange was less intense in fasted than in diabetic rats. Moreover, it should be pointed out that the intramuscular total alanine pool was severely depressed by 48 h-fasting. This could be related to the extensive alanine utilisation in the whole body during the period of fasting before the superfusion experiment. By contrast, a similar increase in total alanine release from muscle of fasted and streptozotocin-diabetic rats (Table 1) was observed. Even though 48 h-fasting had a greater effect on proteolysis than did streptozotocin-diabetes (as reflected by the measurement of net tyrosine release, e. g. 292 ± 25 vs 186 ± 12 nmol tyrosine × g-1 × 2 h-1), the percentage of alanine release originating from proteolysis was the same in fasted muscle as it is in streptozotocin-diabetic extensor digitorum longus muscles (approximatively 60 %). Consequently, leucine contributed to approximately 40 % of alanine a-amino N released in both control and diabetic rats. Only a concomitant increase in proteolysis and de novo synthesis can explain the increase in alanine released from fasted extensor digitorum longus muscles, as previously reported in experimental diabetes [7]. In conclusion, these results confirm the interest of studying both muscle and medium superfusion compartments to better understand alanine regulation within the muscle of fasted rats. Surprisingly, leucine appears less efficient as a donor of a-amino N for the synthesis of alanine in skeletal muscle from fasted than in streptozotocin-diabetic rat even though leucine transamination has been previously described as being increased to a similar extent by fasting or diabetes [8]. These data confirm that alanine synthesis within the muscle may be limited by the availability of amino group acceptors [2]. Whereas a-ketoglurate is added to the medium of superfusion (3 mmol/l) regardless of the animals conditions, glucose is presumably extensively oxidized in superfused extensor digitorum longus muscles from fasted rats, as reflected by the high pH value (7±7.3) in comparison to fed muscles [9]. Consequently, decreased availability of pyruvate may be a limiting factor in glucose-superfused extensor digitorum longus muscles from fasted rats, explaining a lower rate of alanine synthesis in fasted rats than in diabetic rats.

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

Summary To assess the role of leucine as a precursor of alanine α-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 ( ∼ 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. [Diabetologia (1997) 40: 1159–1165]