David G. Gadian

A 31P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) 31P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5-12-times the steady-state rates of ATP utilization (estimated from rates of O2-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a Km for cytosolic ADP of 35 microM and an apparent Vmax of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr in equilibrium ATP or ATP in equilibrium ADP + Pi interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.

The steady-state rate of ATP synthesis in the perfused rat heart measured by 31P NMR saturation transfer

The steady-state rate of ATP synthesis in the isolated, Langendorff-perfused rat heart was determined using a 31P NMR saturation transfer method. At 37°C and a perfusion pressure of 70 cm H2O the value is 2.8 ± 0.3 (n=5 ± S.E.M.) μmol.s−1. (g. dry wt.)−1. The activity of creatine phosphokinase measured in the same experiments was 14.6 ± 1.0 μ mol.s−1 .(g. dry wt.)−1. From the rate of ATP synthesis and the separately measured oxygen consumption we calculated an apparent mitochondrial ADP:O ratio of 3.5 ± 0.8 in the intact tissue.

Studies of metabolism in the isolated, perfused rat heart using 13C NMR.

Since 1974 [I] phosphorus-containing metabolites have been studied in a variety of isolated organs, including skeletal muscle [2], heart [3], liver [4] and kidney [5] using 31P nuclear magnetic resonance (NMR). Recently, non-destructive 13C NMR [8] has been used to define pathways of biosynthesis, measure the partition of isotope flux between different pathways, and determine the displacement of certain cellular reactions from equilibrium in yeast suspensions [6], isolated liver cells [7] and perfused mouse livers [8]. Here we report an NMR investigation of the incorporation of 13C into amino acids in isolated rat hearts perfused with sodium [2-r3C]acetate. Spectra were obtained sufficiently rapidly to follow isotope fluxes and to observe changes in the steady-state levels of enriched metabolites. Our work suggests that the method can be of general use in metabolic studies of the perfused rat heart.

EXAMINATION OF A MYOPATHY BY PHOSPHORUS NUCLEAR MAGNETIC RESONANCE

A 16-year-old boy with myopathy, ophthalmoplegia, and raised basal metabolic rate was examined by the non-invasive technique of phosphorus-31 nuclear magnetic resonance (31 P NMR). The muscles of the forearm showed an abnormal 31P NMR spectrum with a high inorganic phosphate (Pi) content in relation to phosphocreatine (PCr) (PCr/Pi = 4; control = 10). This finding was compatible with the abnormality of mitochondrial function already established by biopsy and offers in addition an explanation for the raised oxygen consumption in this patient. The method of 31P NMR is suited to rapid non-invasive diagnosis in various muscle disorders.

A 31P-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain, and the ionophore R02-2985 (X537A) in the isolated, perfused rat heart

1. Some metabolic effects of increased mechanical activity by the Langendorff-perfused rat heart have been characterized using 31P-NMR. Mechanical activity was increased by infusion of ouabain (0.9-7.0.10(-5) M), the ionophore R02-2985 (1.10(-5) M) or epinephrine (5.10(-8) M). 2. Similar metabolic changes accompanied infusion of each of the positive inotropic agents into hearts perfused with buffer containing 11 mM glucose as the substrate. In each case phosphocreatine concentrations decreased. During the period of epinephrine infusion the phosphocreatine began to recover its original concentration, although there were no significant changes in mechanical activity. 3. Comparisons of the metabolic changes accompanying the positive inotropic and chronotropic effects of epinephrine were made between hearts perfused with either glucose (11 mM), acetate (5 mM) or lactate (5 mM). A time-dependent decrease in phosphocreatine concentrations also accompanied infusion of epinephrine into hearts perfused with lactate as the sole exogenous substrate, but no statistically significant metabolite changes were observed after identical epinephrine infusions with acetate as the substrate. 4. Calculations of the concentration of free ADP assuming equilibrium in the creatine phosphokinase reaction allows estimation of the cytosolic phosphate potential ([ATP]/[ADP][Pi]), which appears to be dependent on the number of factors, including the nature of the exogenous substrate and the level of mechanical activity. 5. Thus, we conclude that there is no general correlation between the phosphate potential and the mitochondrial respiratory rate in the perfused rat heart.

An NMR probe for the study of aerobic suspensions of cells and organelles

Abstract We describe the construction of an NMR probe and cell chamber with good mixing, pH buffering, and oxygenation characteristics, which can be used for relatively dilute cell and organelle suspensions. The 31 P NMR spectra of acceptable signal-to-noise ratios are obtained from approximately 200 mg (protein) of tissues, and kinetic studies of mitochondrial oxidative phosphorylation are demonstrated. Representative spectra from rabbit kidney cortical tubules and rabbit kidney cortical mitochondria are presented.

N-ACETYLASPARTATE AND EPILEPSY

Proton magnetic resonance spectra include signals from N-acetylaspartate, creatine + phosphocreatine, and choline-containing compounds. Abnormalities in these signals can be used in the assessment of patients with intractable epilepsy. In particular, they provide a means of identifying metabolic abnormalities within the temporal lobes, detecting bilateral and diffuse pathology, and aiding lateralization of the seizure focus. The pathology demonstrated on MRS can also be related to cognitive dysfunction.

31P nuclear magnetic resonance of rat pancreatic grafts.

This study investigates whether phosphate metabolite concentrations and intracellular pH alter in early acute rejection of rat pancreatic allografts. In vitro biochemical assays, in vitro 31P nuclear magnetic resonance spectroscopy, and in vivo 31P NMR spectroscopy of the grafts were compared. Duct-ligated, vascularized rat pancreatic isografts and allografts were transplanted onto the infrarenal aorta of the recipients with inferior vena cava venous drainage. In order to obtain reproducible acute rejection, allografting was performed across a major histocompatibility barrier. For the in vitro experiments freeze-clamped graft extracts were prepared and analyzed for adenosine triphosphate concentration by fluorimetry, then placed in an 8.5 Tesla vertical bore magnet. 31P NMR spectra were recorded using a Bruker AM 360 spectrometer operating at 145.7 MHz for 31P. Spectra were acquired from nontransplanted controls; 3-day, 5-day, and 1-month posttransplant isografts, and 3-day and 5-day posttransplant allografts. All grafts examined were functioning satisfactorily. The ATP content of the extracts was significantly lower in the 3- and 5-day allografts than the respective isografts. Invasive in vivo 31P NMR spectra were recorded using surface coils adjacent to the grafts from functioning 5-day posttransplant isografts and allografts (i.e., 3 days prior to an expected elevation in blood sugar from acute rejection in the allografts). The ATP/inorganic phosphate ratios and pH from the in vivo spectra were significantly lower in the allografts than in the isografts. It is concluded that changes in intracellular metabolism occur early in the process of acute rejection and that 31P NMR spectroscopy may provide a means of diagnosing this before current methods.

Nuclear magnetic resonance studies on substrate binding to phosphoglucomutase

Abstract 1. 1. Phosphoglucomutase (α- d -glucose-1,6-bisphosphate: α- d -glucose-1-phosphate phosphotransferase, EC 2.7.5.1) has been assayed by monitoring the proton and phosphorus nuclear magnetic resonances of glucose 1-phosphate and glucose 6-phosphate as a function of time. 2. 2. The activity of phosphoglucomutase in 2 H 2 O is 70% of that in H 2 O. 3. 3. Both anomers of glucose 6-phosphate bind to phosphoglucomutase, although the enzyme is specific to the α-anomer. 4. 4. The Mn 2+ -binding site on phosphoglucomutase is 5.8 A from the phosphorus of bound glucose 6-phosphate. 5. 5. The protons of bound glucose 6-phosphate are about 7–10 A from the Mn 2+ -binding site.

Metabolic studies of whole animals and humans using phosphorus nuclear magnetic resonance

Conclusions31P NMR now provides a totally non-invasive method of studying the metabolism of selected tissues and organs within whole animals and humans. A wide range of studies therefore becomes feasible. For example, there have been problems associated with metabolic studies of isolated organs such as the brain which are particularly sensitive to hypoxia; these problems can now be circumvented by studying the organ within the whole animal. Theories of metabolic control are based largely on studies performed in vitro, and it should now prove possible to test some of these theories directly in vivo. NMR could provide significant advances in our understanding of organ preservation and function. Finally,31P NMR provides a new method of studying the metabolic basis and effects of disease in humans and in animal models. The technique could also provide an objective way of assessing the effectiveness of procedures that are used in the treatment of human disease.