High-resolution nuclear magnetic resonance (NMR) studies of cells and purified mitochondria are discussed to show the kind of information that can be obtained in vivo. In suspensions of Escherichia coli both phosphorus-31 and carbon-13 NMR studies of glycolysis and bioenergetics are presented. In rat liver cells the pathways of gluconeogenesis from carbon-13-labeled glycerol are followed by carbon-13 NMR. In the intact liver cells cytosolic and mitochondrial pH's were separately measured by phosphorus-31 NMR. In purified mitochondria the internal and external concentrations of inorganic phosphate, adenosine diphosphate, and adenosine triphosphate were determined by phosphorus-31 NMR while the pH difference across the membrane was measured simultaneously.

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The gluconeogenic pathway from [1,3-'3C]glycerol has been followed in suspensions of isolated rat hepatocytes at 250C by 13C NMR at 90.5 MHz. The flow of label through the major pathway from glycerol to Lglycerol 3-phosphate and into glucose was Rollowed in cells from control and triiodothyronine-treated rats. Treatment increased the rates of glucose formation and glycerol consumption 2-fold and decreased the aGP level to 40%. We calculate that t60% of the flux is through the mitochondrial glycerol phosphate dehydrogenase in cells from triiodothyronine-treated rats, compared with~15% in cells from the controls. Equal distribution of label between the trioses of glucose was obtained and, because the C3-C4 spin-spin coupling gives the distribution of labeled carbons in the same molecule, it was possible to measure the amount of triose from unlabeled fructose incorporated into the glucose labeled at carbons 1, 3,4, and 6. About 10% of the hexoses had flowed through the pentose cycle and back into the hexose pathway in cells from fasted rats. From the distribution of label at glucose carbons not labeled via the major pathway and from the carbon spin-spin splitting patterns observed, we conclude that transketolase is reversible whereas transaldolase is essentially irreversible in the nonoxidative pentose branch.Recently it has become possible to study metabolism in cellular suspensions by 31P (1) and '3C (2) high-resolution NMR. In recent 31P high-resolution NMR studies of suspensions of rat liver cells we have determined the cytosolic pH and the mitochondrial pH simultaneously from the positions of the two resolved resonances from inorganic phosphate (3). A preliminary account of 13C NMR studies of intermediates and end products of gluconeogenesis in rat liver cells from a '3C-labeled alanine substrate has been reported (4).The present communication illustrates the ability of 13C NMR to follow the gluconeogenic pathway from glycerol in liver cells obtained from euthyroid and hyperthyroid iats and to obtain, very simply, simultaneous quantitative measurements of the contributions of different pathways. This process has been intensively studied in perfused liver (5, 6), freeze-clamped liver (7), and isolated hepatocytes (8,9). The early measurements by Freedland and Krebs (5) of the rate of glucose production from glycerol in perfused rat liver showed that the rate was appreciably increased in thyroxine-treated rats. Even earlier measurements by Lardy and coworkers (10) demonstrated a large increase in the rate of a-glycerophosphate oxidation by liver mitochondria from thyroid-fed rats that was due to the increased activity of the mitochondrial aGP dehydrogenase. METHODS AND MATERIALSLiver parenchymal cells were isolated from male SpragueDawley rats (190-240 g) that had been starved for 24 hr prior to sacrifice. In weight-matched groups, the animals received intraperitoneal injections of either L-3,3',5-triiothyronine (T3; Calbiochem, B grade) dissolved in isotonic saline at pH 9.8 (T3 treated, hyperthyroid)...

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ABSTRACT31P nuclear magnetic relaxation rates for phosphocreatine in intact frog gastrocnemius were compared with those observed in model solutions at 40, a temperature at which muscle maintains its physiological state for at least 5 hr. Both nuclear Overhauser effect and spin-lattice relaxation rate(1/Tj) experiments indicate that dirle-dipole interactions form the dominant relaxation path for P in intact muscle and model solutions, independent of phosphocreatine and Mg concentrations. Spin-spin relaxation rates (1/T2) measured by modified Carr-Purcell-Melboom-Gill spin-echo experiments suggest the importance of scalar coupling modulated by chemical exchange with free Mg. From these results, we estimate the free intracellular Mg in intact muscle as 4.4 mM and demonstrate that 31p T2 experiments can be used as a tool for studying free Mg levels with minimum disturbance of the intact cell. 31P nuclear magnetic resonance (NMR) studies of intact cells to date have concentrated on high-resolution spectra of metabolites in intact muscle (1, 2), erythrocytes (3), yeast (4), and Escherichia coli and Ehrlich ascites tumor cells (5, 6). We report here on the feasibility of measuring the relaxation parameters of phosphocreatine (PCr) in intact frog muscle at 4°. The spin-lattice relaxation time (TI) and the spin-spin relaxation time (T2) of PCr in intact muscle are compared with those observed in model solutions at 4°. Both relaxation rates are valuable as dynamic probes of the internal environment in muscle because T2 can be sensitive to processes that do not affect T, at all. These relaxation studies also enable us to assign a value for the free intracellular Mg concentration in intact gastrocnemius via an intrinsically nonintrusive method.It is now clear that metal ions are of central importance in many biological reactions and knowledge of free Mg levels has particular significance. For (10). Spinning 10-mm samples were used; the field was locked either to a capillary of D20 (muscles and PCr/H20 solutions) or D20 solvent. Typical conditions included a 900 pulse of ca 20 lisec, 32-128 scans of 4000 data points each, sweep width of 1 kHz, exponential multiplication introducing 0.6-Hz line broadening, and 'H irradiation.T, measurements were made with the following pulse sequence: 900 pulse, spoil field homogeneity, wait delay time r, 90°pulse, sample free-induction decay, and then spoil field homogeneity. This sequence (11), which was repeated n times, afforded a considerable saving of time over the inversion-recovery method in the T, regimen of 31P in PCr, thus making it possible to carry out T, measurements on muscle during the interval in which there was no more than a 10% change in PCr.The Carr-Purcell-Meiboom-Gill (CPMG) (12) T2 measurements were performed on a Bruker B-KR pulsed spectrometer operating in conjunction with the same Nicolet data system and a 21.14 kG magnet used for high-resolution spectra. Two home-built modifications to the probe of this pulsed spectrometer were made so that satisfactory results ...