George K. Radda

Oxygenation dependence of the transverse relaxation time of water protons in whole blood at high field.

At high and medium magnetic field, the transverse NMR relaxation rate (T-1(2)) of water proteins in blood is determined predominantly by the oxygenation state of haemoglobin. T-1(2) depends quadratically on the field strength and on the proportion of haemoglobin that is deoxygenated. Deoxygenation increases the volume magnetic susceptibility within the erythrocytes and thus creates local field gradients around these cells. From volume susceptibility measurements and the dependence of T-1(2) on the pulse rate in the Carr-Purcell-Meiboom-Gill experiment, we show that the increase in T-1(2) with increasing blood deoxygenation arises from diffusion of water through these field gradients.

Abnormal Cardiac and Skeletal Muscle Energy Metabolism in Patients With Type 2 Diabetes

Background It is well known that patients with type 2 diabetes have increased risk of cardiovascular disease, but it is not known whether they have underlying abnormalities in cardiac or skeletal muscle high‐energy phosphate metabolism. Methods and Results We studied 21 patients with type 2 diabetes with no evidence of coronary artery disease or impaired cardiac function, as determined by echocardiography, and 15 age‐, sex‐, and body mass index‐matched control subjects. Cardiac high‐energy phosphate metabolites were measured at rest using 31P nuclear magnetic resonance spectroscopy (MRS). Skeletal muscle high‐energy phosphate metabolites, intracellular pH, and oxygenation were measured using 31P MRS and near infrared spectrophotometry, respectively, before, during, and after exercise. Although their cardiac morphology, mass, and function appeared to be normal, the patients with diabetes had significantly lower phosphocreatine (PCr)/ATP ratios, at 1.50±0.11, than the healthy volunteers, at 2.30±0.12. The cardiac PCr/ATP ratios correlated negatively with the fasting plasma free fatty acid concentrations. Although skeletal muscle energetics and pH were normal at rest, PCr loss and pH decrease were significantly faster during exercise in the patients with diabetes, who had lower exercise tolerance. After exercise, PCr recovery was slower in the patients with diabetes and correlated with tissue reoxygenation times. The exercise times correlated negatively with the deoxygenation rates and the hemoglobin (Hb)A1c levels and the reoxygenation times correlated positively with the HbA1c levels. Conclusions Type 2 diabetic patients with apparently normal cardiac function have impaired myocardial and skeletal muscle energy metabolism related to changes in circulating metabolic substrates. (Circulation. 2003;107:3040‐3046.)

Physical training improves skeletal muscle metabolism in patients with chronic heart failure

OBJECTIVES This study investigated the effects of physical training on skeletal muscle metabolism in patients with chronic heart failure. BACKGROUND Skeletal muscle metabolic abnormalities in patients with chronic heart failure have been associated with exercise intolerance. Muscle deconditioning is a possible mechanism for the intrinsic skeletal muscle metabolic changes seen in chronic heart failure. METHODS We used phosphorus-31 nuclear magnetic resonance spectroscopy to study muscle metabolism during exercise in 12 patients with stable ischemic chronic heart failure undergoing 8 weeks of home-based bicycle exercise training in a randomized crossover controlled trial. Changes in muscle pH and concentrations of phosphocreatine and adenosine diphosphate (ADP) were measured in phosphorus-31 spectra of calf muscle obtained at rest, throughout incremental work load plantar flexion until exhaustion and during recovery from exercise. Results were compared with those in 15 age-matched control subjects who performed a single study only. RESULTS Before training, phosphocreatine depletion, muscle acidification and the increase in ADP during the 1st 4 min of plantar flexion exercise were all increased (p < 0.04) compared with values in control subjects. Training produced an increase (p < 0.002) in incremental plantar flexion exercise tolerance. After training, phosphocreatine depletion and the increase in ADP during exercise were reduced significantly (p < 0.003) at all matched submaximal work loads and at peak exercise, although there was no significant change in the response of muscle pH to exercise. After training, changes in ADP were not significantly different from those in control subjects, although phosphocreatine depletion was still greater (p < 0.05) in trained patients than in control subjects. The phosphocreatine recovery half-time was significantly (p < 0.05) shorter after training, although there was no significant change in the half-time of adenosine diphosphate recovery. In untrained subjects, the initial rate of phosphocreatine resynthesis after exercise (a measure of the rate of oxidative adenosine triphosphate [ATP] synthesis) and the inferred maximal rate of mitochondrial ATP synthesis were reduced compared with rates in control subjects (p < 0.003) and both were significantly increased (p < 0.05) by training, so that they were not significantly different from values in control subjects. CONCLUSIONS The reduction in phosphocreatine depletion and in the increase in ADP during exercise, and the enhanced rate of phosphocreatine resynthesis in recovery (which is independent of muscle mass) indicate that a substantial correction of the impaired oxidative capacity of skeletal muscle in chronic heart failure can be achieved by exercise training.

Skeletal muscle metabolism in patients with congestive heart failure: relation to clinical severity and blood flow.

We and others have previously demonstrated excessive phosphocreatine (PCr) depletion and acidosis in skeletal muscle during exercise in patients with congestive heart failure (CHF). In the present study, we performed serial measurements of PCr and pH during gradually incremental flexor digitorum superficialis exercise in 22 patients with CHF and 11 age-matched controls to determine: (1) whether abnormalities were present at the same relative workloads (a comparison that would at least partially compensate for differences in muscle mass), (2) the temporable course of the metabolic changes, (3) the relationship of the metabolic findings to clinical variables, and (4) the relationship of the metabolic abnormalities to forearm blood flow. The patients with CHF had significantly lower [PCr] and pH at all submaximal levels of exercise, and these abnormalities were apparent from the onset of low-level exercise. There was considerable heterogeneity among the patients with CHF with respect to the metabolic findings, with 14 of 22 exhibiting either PCr or pH values more than 2 SDs below normal. Patients whose capacity was more limited during the protocol had lower [PCr], and especially pH, at low loads than did other patients with CHF or the control subjects. The more symptomatic patients and those with more limited bicycle exercise tolerance also had lower pH values. In contrast, there were no significant differences in forearm blood flow between the patients and controls and no relationship between forearm blood and either clinical variables or the metabolic findings. These results indicate that skeletal muscle metabolic abnormalities are present in many patients with CHF and that they are not primarily due to either muscle atrophy or impaired blood flow. These changes may explain in part the marked heterogeneity of symptom status and exercise capacity of patients with similar degrees of cardiac dysfunction.

Uncoupling proteins in human heart

Abnormal energetic activity in heart failure correlates inversely with plasma free-fatty-acid concentrations. However, the link between energetic and metabolic abnormalities is unknown. To investigate this association, we obtained blood samples from 39 patients undergoing coronary artery bypass graft surgery. Patients fasted overnight before samples were taken. When plasma free-fatty-acid concentrations were raised, cardiac mitochondrial uncoupling proteins (UCP) increased (isoform UCP2, p<0.0001; isoform UCP3, p=0.0036) and those of glucose transporter (GLUT4) protein decreased (cardiac, p=0.0001; skeletal muscle, p=0.0006). Consequently, energy deficiency in heart failure might result from increased mitochondrial UCPs (ie, less efficient ATP synthesis) and depleted GLUT4 (ie, reduced glucose uptake). New treatment to correct these energy defects would be to simultaneously lower plasma free fatty acids and provide an alternative energy source.

Insulin, ketone bodies, and mitochondrial energy transduction.

Addition of insulin or a physiological ratio of ketone bodies to buffer with 10 mM glucose increased efficiency (hydraulic work/energy from O2 consumed) of working rat heart by 25%, and the two in combination increased efficiency by 36%. These additions increased the content of acetyl CoA by 9‐ to 18‐fold, increased the contents of metabolites of the first third of the tricarboxylic acid (TCA) cycle 2‐ to 5‐fold, and decreased succinate, axaloacetate, and aspartate 2‐ to 3‐fold. Suc‐ cinyl CoA, fumarate, and malate were essentially unchanged. The changes in content of TCA metabolites resulted from a reduction of the free mitochondrial NAD couple by 2‐ to 10‐fold and oxidation of the mitochondrial coenzyme Q couple by 2‐ to 4‐fold. Cytosolic pH, measured using 31P‐NMR spectra, was invariant at about 7.0. The total intracellular bicarbonate indicated an increase in mitochondrial pH from 7.1 with glucose to 7.2, 7.5, and 7.4 with insulin, ketones, and the combination, respectively. The decrease in Eh7 of the mitochondrial NAD couple, Eh7NAD∗/NADH, from ‐280 to ‐300 mV and the increase in Eh7 of the coenzyme Q couple, Eh7Q/QH2, from ‐4 to +12 mV was equivalent to an increase from ‐53 kJ to ‐60 kJ/2 mol e in the reaction catalyzed by the mitochondrial NADH dehydrogenase multienzyme com‐plex (EC 1.6.5.3). The increase in the redox energy of the mitochondrial cofactor couples paralleled the increase in the free energy of cytosolic ATP hydrolysis, ΔGatp‐ The potential of the mitochondrial relative to the cytosolic phases, Emito/cyto, calculated from ΔGatp and ΔpH on the assumption of a 4 H+ transfer for each ATP synthesized, was ‐143 mV during perfusion with glucose or glucose plus insulin, and decreased to ‐120 mV on addition of ketones. Viewed in this light, the moderate ketosis characteristic of prolonged fasting or type II diabetes appears to be an elegant compensation for the defects in mitochondrial energy transduction associated with acute insulin deficiency or mitochondrial senescence.—Sato, K., Kashiwaya, Y., Keon, C. A., Tsuchiya, N., King, M. T., Radda, G. K., Chance, B., Clarke, K., Veech, R. L. Insulin, ketone bodies, and mitochondrial energy transduction. FASEB J. 9, 651‐658 (1995)

In vivo assessment of pyruvate dehydrogenase flux in the heart using hyperpolarized carbon-13 magnetic resonance.

The advent of hyperpolarized 13C magnetic resonance (MR) has provided new potential for the real-time visualization of in vivo metabolic processes. The aim of this work was to use hyperpolarized [1-13C]pyruvate as a metabolic tracer to assess noninvasively the flux through the mitochondrial enzyme complex pyruvate dehydrogenase (PDH) in the rat heart, by measuring the production of bicarbonate (H13CO3−), a byproduct of the PDH-catalyzed conversion of [1-13C]pyruvate to acetyl-CoA. By noninvasively observing a 74% decrease in H13CO3− production in fasted rats compared with fed controls, we have demonstrated that hyperpolarized 13C MR is sensitive to physiological perturbations in PDH flux. Further, we evaluated the ability of the hyperpolarized 13C MR technique to monitor disease progression by examining PDH flux before and 5 days after streptozotocin induction of type 1 diabetes. We detected decreased H13CO3− production with the onset of diabetes that correlated with disease severity. These observations were supported by in vitro investigations of PDH activity as reported in the literature and provided evidence that flux through the PDH enzyme complex can be monitored noninvasively, in vivo, by using hyperpolarized 13C MR.

NMR studies on phospholipid bilayers. Some factors affecting lipid distribution

1. 1H-NMR and 31P-NMR are used to measure the outside/inside distribution of phospholipids in mixed vesicles. 2. Ferricyanide is a suitable shift reagent for measuring the outside/inside ratio of lecithin using 1H-NMR even when the phospholipid mixture contains negative lipids. 3. 31P-NMR can be used to measure the distribution of all phospholipids present provided the resonances are separated. 4. At 36.4 MHz the inside and outside phosphorus in lecithin vesicles have different chemical shifts. The separation at room temperature is 4-5 Hz and the individual linewidths are about 4Hz. 5. In a mixture of lecithin with phosphatidylethanolamine the latter has preference for the inside layer of the bilayer. The same holds for mixtures of lecithin with phosphatidylserine, phosphatidylinositol and phosphatidic acid. 6. In mixtures of lecithin and phosphatidylserine the preference of the latter for the inside is increased at lower pH under which conditions the negative charge of the phosphatidylserine is decreased. 7. In mixtures of lecithin with sphingomyelin the lecithin has a higher concentration at the inside. 8. The effect of vesicle size on the 31P-NMR linewidth and the temperature dependence of this linewidth is in agreement with the conclusion of Berden et al. (FEBS Lett. (1974), 46, 55-58) that the chemical shift anisotropy, modulated by the isotropic tumbling of the vesicles, makes a contribution to the linewidth. The chemical shift difference between outside and inside phosphorus can be used as a parameter for the measurement of the packing density at the inside and of the size of the vesicles. 9. It is concluded that both charge and the packing properties of the head group are major factors in determining the distribution of phospholipids in mixed vesicles.

Differential scanning calorimetry and 31P NMR studies on sonicated and unsonicated phosphatidylcholine liposomes

1. Phase transitions in sonicated (vesicles) and unsonicated liposomes composed of various synthetic phosphatidylcholines are monitored using differential scanning calorimetry and 31P NMR. 2. The temperature (Tc), heat content and width of the phase transition are comparable in both vesicles and liposomes prepared from 1,2-dipalmitoyl phosphatidylcholine and 1,2-dimyristoyl phosphatidylcholine. In vesicles composed of a (1 : 1) mixture of 1,2-dipalmitoyl phosphatidylcholine and 1,2-dioleoyl phosphatidylcholine phase separation occurs as in the bilayers of the unsonicated liposomes. 3. The linewidth of the 31P resonances in vesicles is not greatly dependent upon the fatty acid composition when the lipids are in the disordered liquid crystalline state (above Tc). When the lipids are in the gel state (below Tc), however, there is a marked increase in linewidth, demonstrating a reduction in motion of the phosphate group. 4. The ratio of the amounts of phosphatidylcholine present in the outside and inside monolayter of the vesicle membrane was determined with 31P NMR using Nd3+ as a non-permeating shift reagent. 5. The outside/inside ratio is dependent upon the hydrocarbon chain length. Increasing chain length gives a lower outside/inside ratio and a larger vesicle. Introduction of cis or trans double bonds in the chain influences the outside/inside ratio slightly. 6. The incorporation of cholesterol decreases the outside/inside ratio and increases the size of 1,2-dimyristoyl phosphatidylcholine vesicles. The cholesterol concentration in the outside and inside monolayer is approximately the same. The size of the 1,2-dioleoyl phosphatidylcholine vesicles is also increased by cholesterol incorporation but the outside/inside distribution is also increased, especially between 30 and 50 mol% cholesterol. In these vesicles cholesterol is asymmetrically distributed and strongly prefers the inside monolayer of the vesicle.

Real-time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy

The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart disease. In this study, we measured Krebs cycle flux in real time in perfused rat hearts using hyperpolarized magnetic resonance spectroscopy (MRS). [2‐13C]Pyru‐ vate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of 13C‐labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of 13C‐ labeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly elevated. These results showed that the control and fluxes of the Krebs cycle in heart disease can be studied using hyperpolarized [2‐13C]pyruvate.— Schroeder, M. A., Atherton, H. J., Ball, D. R., Cole, M. A., Heather, L. C., Griffin, J. L., Clarke, K., Radda, G. K., Tyler, D. J. Real‐time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy. FASEBJ. 23, 2529–2538 (2009)