The C-13-1 NMR peak in proton-decoupled spectra of liver glycogen solution was quantitatively analyzed by three types of model-function fitting algorithms: iterative line-fitting in the frequency domain (MDCON); iterative least-squares fitting (VARPRO) in the time domain; and noniterative singular value decomposition-based analysis (HTLS), also in the time domain, Quantification results were compared with manual integration values, Performance of the algorithms was tested at different signal-to-noise ratios (S/N) of the glycogen C-1 peak, This was achieved by varying the number of scans summed prior to analysis, Since T-2 relaxation in glycogen has been shown to be multiexponential [Overloop, K. et al. Magn, Reson. Med. 36, 45-51 (1996], the exact quantification of the C-1 glycogen signal requires a model function comprising a sum of Lorentzian components, each with a different broadening at the glycogen frequency, This paper focuses on the performances of the above methods to fit such a multicomponent resonance line, In the frequency domain, line fitting with two Lorentz lines gives good results at sufficiently high S/N, In the time domain, VARPRO performs better than HTLS because fixed values can be imposed to the linewidth of the components at the common C-1 frequency, thereby reducing convergence problems at low S/N. (C) 1997 by John Wiley & Sons, Ltd.

This study is the first report on the multiexponential T 2 relaxation of the 13 C‐1 carbon of glycogen. In contrast to T 1 relaxation, which does not display observable multiexponential decay behavior, T 2 relaxation is described by a continuous distribution of T 2 times. Changes in molecular weight and sample viscosity, which affect the overall mobility of the glycogen particle have little influence on T 1 and T 2 relaxation times. This is in contradiction with earlier results that T 2 is dominated by the overall motion of the glycogen particles [L.‐H. Zang Biochemistry 29, 6815–6820 (1990)]. T 1 depends strongly on the external field B o and is almost temperature independent in the range 23–37°C whereas T 2 is field independent and varies appreciably with temperature. The experimental T 1 and T 2 relaxation data are shown to be consistent with existing theoretical models for relaxation, suitably modified to include a distribution of correlation times for the internal motions. The presence of fast decaying components (short T 2 ) in the FID implies broad line components in the frequency spectrum and the corresponding need to appropriately set the integration limits for the quantification of the glycogen peak.

In search for a nonmetabolized, superior glucose analogue to study the mechanism of glucose-induced glycogen synthesis, we have tested 2-deoxy-2-fluoro-alpha-D-glucopyranosyl fluoride, which inhibits muscle phosphorylase b 10 fold better than does glucose (Street, I. P., Armstrong, C. R., and Withers, S. G. (1986) Biochemistry 25, 6021-6027), In a gel-filtered liver extract, 0.6 mM analogue and 10 mM glucose equally accelerated the inactivation of phosphorylase and shortened the latency before the activation of glycogen synthase, The analogue was not measurably defluorinated or phosphorylated by intact hepatocytes, as monitored by F-19 NMR. When added to isolated hepatocytes, 10 mM analogue inactivated phosphorylase more extensively than did 50 mM glucose, but unlike glucose, it did not result in the activation of glycogen synthase, Therefore, the binding of glucose to phosphorylase a can account for the inactivation of phosphorylase, but the metabolism of glucose (probably to Glc-6-P) appears to be required to achieve activation of glycogen synthase.The livers of overnight-fasted, anesthetized mice contained appreciable amounts of both phosphorylase a and glycogen synthase a, without net glycogen accumulation, Likewise, hepatocytes isolated from fasted rats and incubated with 10 mM glucose contained 41% of phosphorylase and 32% of glycogen synthase in the a form, and these values remained stable for 1 h, while glycogen accumulated at only 22% of the rate expected from the glycogen synthase activity, The addition of 10 mM analogue decreased phosphorylase a to 10% without significant change in glycogen synthase a (38%), but with a 4-fold increased rate of glycogen accumulation. These findings imply that synthase a is fully active in the liver of the fasted animal and that the absence of net glycogen synthesis is due to continuous glycogenolysis by phosphorylase a.

In search for a nonmetabolized, superior glucose analogue to study the mechanism of glucose-induced glycogen synthesis, we have tested 2-deoxy-2-fluoro-α-D-glucopyranosyl fluoride, which inhibits muscle phosphorylase b 10-fold better than does glucose (Street, I. P., Armstrong, C. R., and Withers, S. G. (1986) Biochemistry 25, 6021-6027). In a gel-filtered liver extract, 0.6 mM analogue and 10 mM glucose equally accelerated the inactivation of phosphorylase and shortened the latency before the activation of glycogen synthase. The analogue was not measurably defluorinated or phosphorylated by intact hepatocytes, as monitored by F NMR. When added to isolated hepatocytes, 10 mM analogue inactivated phosphorylase more extensively than did 50 mM glucose, but unlike glucose, it did not result in the activation of glycogen synthase. Therefore, the binding of glucose to phosphorylase a can account for the inactivation of phosphorylase, but the metabolism of glucose (probably to Glc-6-P) appears to be required to achieve activation of glycogen synthase. The livers of overnight-fasted, anesthetized mice contained appreciable amounts of both phosphorylase a and glycogen synthase a, without net glycogen accumulation. Likewise, hepatocytes isolated from fasted rats and incubated with 10 mM glucose contained 41% of phosphorylase and 32% of glycogen synthase in the a form, and these values remained stable for 1 h, while glycogen accumulated at only 22% of the rate expected from the glycogen synthase activity. The addition of 10 mM analogue decreased phosphorylase a to 10% without significant change in glycogen synthase a (38%), but with a 4-fold increased rate of glycogen accumulation. These findings imply that synthase a is fully active in the liver of the fasted animal and that the absence of net glycogen synthesis is due to continuous glycogenolysis by phosphorylase a.

BACKGROUND:Experiments were carried out using the new Na(+)-H+ exchange inhibitor (3-methylsulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate (HOE 694) to assess the role of Na(+)-H+ exchange in myocardial ischemic and reperfusion injury.METHODS AND RESULTS:Three groups of rabbit hearts (n = 5 in each) were perfused with blood and were subjected to 45 minutes of global normothermic (37 degrees C) ischemia, followed by 1 hour of reperfusion. Group 1 was the control group (vehicle only); in group 2, HOE 694 (1 mumol/L) was administered before ischemia (pretreatment group); and in group 3, HOE 694 was given only during reperfusion to separate actions exerted during ischemia from those specifically obtained during reperfusion. End-diastolic pressure rise at 1 hour of reperfusion was reduced by administration of HOE 694 starting before ischemia (from 52.2 +/- 8.5 mm Hg in group 1 to 17.6 +/- 4.5 mm Hg in group 2, P < .01) or starting on reperfusion (28.8 +/- 5.4 mm Hg in group 3, P < .05 versus group 1). Left ventricular developed pressure (LVDP) and its derivative (dP/dt) recovered better in HOE 694-pretreated hearts (LVDP, 79 +/- 9.9 mm Hg in group 2 versus 24.8 +/- 10 mm Hg in group 1; dP/dt, 1580 +/- 198 mm Hg/s versus 340 +/- 221 mm Hg/s, P < .01). In hearts treated only on reperfusion, some improvement was observed, which, however, did not reach statistical significance. Coronary flow on reperfusion was higher in groups 2 and 3 compared with controls, and no "no-reflow" was observed. Two additional groups of hearts were perfused with phosphate-free Krebs-Henseleit solution to enable studies with 31P nuclear magnetic resonance (NMR). ATP was better preserved in HOE 694-pretreated (62 +/- 4.9% of preischemic value) than in control hearts (44 +/- 3.3%) at the end of 30 minutes of reperfusion, and phosphocreatine resynthesis was higher (109 +/- 3.7% versus 86 +/- 5.4%). HOE 694 did not affect the time course of intracellular acidosis during ischemia but suppressed a small alkaline overshoot occurring early in reperfusion (pH 6.96 +/- 0.02 in HOE 694-pretreated hearts versus 7.14 +/- 0.05 in control hearts). Electron microscopy with Ca2+ staining of the blood-perfused hearts showed that clumping of Ca2+ aggregates in mitochondria was prevented by HOE 694.CONCLUSIONS:Postischemic dysfunction was associated with a rise in end-diastolic pressure. This rise was effectively blocked by HOE 694. The drug was most effective when hearts were treated before ischemia, although partial protection was observed when administration was started on reperfusion. The action of HOE 694 strengthens the idea that Na(+)-H+ exchange during both ischemia and reperfusion contributes to contractile dysfunction.

31P-NMR spectroscopy has become the major tool for studying myocardial high energy phosphates. Conflicting results concerning NMR visibility of ATP in ischaemic myocardium were reported. A detailed study was undertaken to resolve this controversy. After cardioplegic arrest, canine hearts were excised and preserved for 24 h at 1°C (group 1) or for 6 h at 23°C (group 2). ATP breakdown was followed by31P-NMR spectroscopy in a transmural piece of the anterior wall introduced in the NMR magnet, and by HPLC analysis using serial transmural biopsies from the rest of the anterior wall. At both temperatures, identical relative ATP decay curves were obtained, whether measured by NMR or by HPLC. Absolute quantification of ATP was carried out after varying periods of ischaemia at 1°C. The NMR-measured ATP concentration was 106±8% of the ATP concentration determined by HPLC. From our experiments, we conclude that ATP visibility for31P-NMR spectroscopy is complete and constant during prolonged periods of hypothermic ischaemia in canine hearts.

Previous investigations concerning the NMR relaxation in adsorbed water show that both the longitudinal and the transverse proton magnetization decay in a multiexponential way. Two or more components can always be distinguished in those decay curves. We propose a model for adsorbed water which can account for these different relaxation components and with the aid of this model we give a quantitative description of the longitudinal relaxation in water adsorbed on two silica gels and two controlled pore glasses. The model assumes three phases:the protons of the OH groups on the adsorbent surface, the protons of the water molecules close to the surface ("bound" water), and the protons in the rest of the water ("free" water). We prove the occurrence of cross relaxation between the OH protons and the protons in the bound water and of exchange between bound and free water. The OH protons have very slow longitudinal relaxation with T1 = 4-5 s and very fast transverse relaxation with a characteristic time of about 40 μs. The amount of OH protons corresponds to six OH groups per 100 Å2 for all adsorbents studied. We show that the influence of the surface on the longitudinal relaxation of the adsorbed water is limited to one adsorbed layer, that the longitudinal relaxation time for the bound water lies between 20 and 60 ms, and that the free water exhibits a longitudinal relaxation time comparable to the value for pure water (3.3 s). Values for the cross-relaxation times and exchange times are deduced.

We report on the 13C NMR visibility of the C‐1 glycosidic carbon of α‐particulate glycogen in perfused rat liver. We used rats fed ad libitum, animals refed after a 48 h fast with a sucrose supplement with or without glucocorticoid treatment, and gsd/gsd rats with a hepatic glycogen storage disease due to phosphorylase kinase deficiency. Thus we studied a wide range of glycogen levels (25‐140 mg/g liver). All livers were perfused with 15 mM glucose, to maintain constant glycogen levels. Failure to activate glycogen phosphorylase ensures stable glycogen levels in gsd/gsd livers. Natural abundance 13C NMR signals were calibrated against a phantom containing a fixed amount of glycogen. Accumulated free induction decays were analysed after Fourier transformation by numerical integration, or by direct analysis of the signal in the time domain using a non‐iterative method based on singular value decomposition. NMR quantification of the glycogen correlated well with the chemical determination over the whole concentration range. However, the precision (reproducibility) of glycogen determinations (be it by chemical methods or by NMR spectroscopy) may pose problems. Authors should be encouraged to report systematically on the precision of their methods.

The freezing of water adsorbed on high-surface-area materials such as silica gel, controlled-pore glass, and activated charcoal is investigated with NMR methods. Part of the pore water freezes at the temperature predicted by the Kelvin equation and the rest of the water does not freeze in the sense that it does not assume the structure of ice on cooling below this freezing point. This bound water exhibits a distribution of correlation times, and information about the width of this distribution is obtained. Values for the activation enthalpy of the bound water are also deduced. An alternative method for the determination of the pore volume of an adsorbent is proposed and it is shown that. for adsorbents with small pores (r < 100 Å), this method results in a much better estimate for the total pore volume than the more common mercury-intrusion method.

The NMR relaxation behavior of the proton magnetization in adsorbed water is investigated. Longitudinal as well as transverse relaxation of the adsorbed water as a whole shows a distinct nonexponentiality and the nature of this nonexponentiality is studied. Different methods of fitting distribution of relaxation times are examined and various simulated and experimental decay curves are submitted to these fitting methods. The results of these fittings clearly indicate that both the longitudinal and the transverse relaxation of the proton magnetization in adsorbed water is related to two or three discrete components and that there is certainly no broad distribution of relaxation times present in these water-adsorbent systems.

Article Proton NMR Study of the Different Water Systems in Moistened Spores of Phycomyces Blakesleeanus* was published on January 1, 1987 in the journal Zeitschrift für Physikalische Chemie (volume 153, issue 1-2).

The distribution and properties of the cellular water in sporangiospores of Phycomyces blakesleeanus were investigated using proton-NMR. In dormant spores different classes of water were characterized by a difference in their transverse relaxation times (T 2). The amount of cytoplasmic water was estimated to be as low as about 700 mg (g dry wt)−1 and its small T 2 (18·2 ms) indicated a very limited mobility. About 10 min after induction of germination (by a heat shockor by addition of 0·1 m-acetate), both the content and the mobility of the cytoplasmic water increased sharply. These changes coincided with a rapid breakdown of most of the cellular trehalose and with the production (and leakage from the spores) of large amounts of glycerol. The role of these biochemical changes is discussed in relation to the water status of the spores.