13C-NMR: a simple yet comprehensive method for analysis of intermediary metabolism.

13C-NMR is a particularly attractive tool for metabolic studies because of its inherent simplicity: all labeled products at sufficient concentration may be identified and analysed in a single spectrum. However, the real power behind the approach presented here is the ability to measure groups of individual 13C-isotopomers (isotope isomers). The information that this provides is superior to conventional tracer techniques, allowing a very detailed description of metabolic events. Several examples are given of the value of this convenient and straightforward analysis for some problems of current interest in intermediary metabolism.

Effects of amino acids on substrate selection, anaplerosis, and left ventricular function in the ischemic reperfused rat heart.

The effect of aspartate and glutamate on myocardial function during reperfusion is controversial. A beneficial effect has been attributed to altered delivery of carbon into the citric acid cycle via substrate oxidation or by stimulation of anaplerosis, but these hypotheses have not been directly tested. 13C isotopomer analysis is well suited to the study of myocardial metabolism, particularly where isotopic and metabolic steady state cannot be established. This technique was used to evaluate the effects of aspartate and glutamate (amino acids, AA) on anaplerosis and substrate selection in the isolated rat heart after 25 min of ischemia followed by 30 or 45 min of reperfusion. Five groups of hearts (n = 8) provided with a mixture of [1,2-13C]acetate, [3-13C]lactate, and unlabeled glucose were studied: control, control plus AA, ischemia followed by 30 min of reperfusion, ischemia plus AA followed by 30 min of reperfusion, and ischemia followed by 45 min of reperfusion. The contribution of lactate to acetyl-CoA was decreased in postischemic myocardium (with a significant increase in acetate), and anaplerosis was stimulated. Metabolism of 13C-labeled aspartate or glutamate could not be detected, however, and there was no effect of AA on functional recovery, substrate selection, or anaplerosis. Thus, in contrast to earlier reports, aspartate and glutamate have no effect on either functional recovery from ischemia or on metabolic pathways feeding the citric acid cycle.

Effect of fasting and acute ethanol administration on the energy state of in vivo liver as measured by 31P-NMR spectroscopy.

The effects of 48 h fasting, administration of ethanol or 2,4-dinitrophenol, on the phosphorus-containing metabolites in liver in vivo have been determined utilizing 31P nuclear magnetic resonance spectroscopy. These measurements were combined with determinations of metabolite concentrations in livers which were freeze-clamped immediately after the NMR measurements were completed. Administration of sub-lethal amounts of dinitrophenol dramatically decreased ATP and increased Pi concentrations in liver in vivo as indicated by a 2.7-fold increase in the NMR-derived [Pi]/[ATP] ratio. Ethanol administration to fed animals increased the NMR-derived [Pi]/[ATP] ratio 27%; in contrast, the same amount of ethanol administered to fasted animals decreased the NMR-derived [Pi]/[ATP] ratio 30%. The NMR visible Pi and ADP represent about 50% and 15% of the total Pi and ADP, respectively. The phosphorylation potentials calculated from the NMR visible Pi and ADP were an order of magnitude higher than those obtained from metabolite concentrations in freeze-clamped tissue. There was no apparent correlation between the phosphorylation potentials derived from either the NMR spectral analyses or from metabolite concentrations and the hepatic [NAD+]/[NADH] ratio. The chemical shift of Pi indicated that ethanol administration elicited a decrease in pH of 0.1 unit in liver in vivo. Hepatic free [Mg2+] was increased 21% in fasted animals, but was unaffected by ethanol administration.

The metabolic state of the rat liver in vivo measured by 31P-NMR spectroscopy.

Previous 31P nuclear magnetic resonance (NMR) studies have measured the concentrations of phosphates, free Mg2+, pH and flux through enzyme-catalyzed reactions in a variety of tissues. A surgically-implanted coil has been developed to measure these parameters in the rat liver in vivo, and to assess the effect of external perturbations on the concentrations and physiological environment of phosphorus metabolities in the liver. The sensitive volume and optimal pulse were determined for the coil, which was insulated to exclude signal from surrounding tissues. The metabolic stability of the liver during acquisition of spectra was demonstrated by normal values for [Pi], [ATP], [lactate], and [pyruvate] in livers which were freeze-clamped immediately after completion of the NMR experiment. The stability was also confirmed by constant values for intracellular pH (7.2), free [Mg2+] (0.7 mM), and NMR detectable [Pi]/[ATP]. The sensitivity of the 31P-NMR spectrum of the liver in vivo to the physiological state of the animals was illustrated by comparing spectra from fed and 48 h fasted rats. The major qualitative differences were an increase in the pyridine nucleotide/adenine nucleotide ratio, and a small, but consistent shift in the frequency of the composite phosphomonoester peak. The spin-lattice relaxation time of each major phosphate resonance was measured in vivo using a modified homospoil saturation recovery pulse sequence; the T1 of ATP gamma-phosphate was 0.17 s. Selective saturation experiments did not detect magnetization transfer between the ATP gamma-phosphate and inorganic phosphate.

The determination of magnesium in human blood plasma by 31P magnetic resonance spectroscopy using a macrocyclic reporter ligand.

The ligand 1,4,7-triazacyclononane-1,4,7-tris(methylene methylphosphinic acid), NOTMP, was used to measure free MgII levels in blood plasma by 31P MRS. Separate resonances were observed for the free ligand and the MgII complex and the ratio of their resonance areas was used to evaluate the free, ionized MgII concentration, [Mg]free. The CaII and the ZnII complexes gave rise to separate resonances in the 31P spectrum in an aqueous sample. In human blood plasma samples, however, these resonances were never observed thus excluding the interference of these metal ions. Heparin, up to 150 units/ml, had no influence on the Mg-NOTMP equilibrium. The 31P MRS methodology was applied to twenty human blood plasma samples. Total MgII ([Mg]total), as measured by atomic absorption spectroscopy, averaged 0.85 +/- 0.12 mM while free ionized MgII ([Mg]free) measured by 31P MRS was 0.66 +/- 0.09 mM. The 31P MRS method gave inherently larger values for free ionized MgII than that reported by ion-selective electrodes (ISE). This was traced to a redistribution of existing plasma MgII species after the addition of about 2 mM of NOTMP. Calculations using existing thermodynamic data show that the ionized MgII concentration (iMg) and the concentration of MgII weakly complexed to small anions (Mg(comp)) both drop after the addition of NOTMP, with Mg(comp) dropping to negligible levels. Thus, the 31P MRS method appears to be less sensitive to variations in the concentration of weakly binding anions (bicarbonate, carbonate, chloride, lactate, phosphate, etc.) than the ISE method. Our data indicates that the difference between Mg(total), as measured by atomic absorption spectroscopy, and Mg(free), as measured by 31P MRS, provides an direct estimate of the protein bound MgII fraction.

Nuclear magnetic resonance imaging in Marfan's syndrome.

Detection and evaluation of aortic root and other cardiovascular abnormalities in patients with Marfan's syndrome are important in determining appropriate therapy and preventing premature mortality. To evaluate the role of nuclear magnetic resonance imaging (NMR) in this syndrome, 10 patients were evaluated using a 0.35 tesla commercial nuclear magnetic resonance imaging system. Findings from these studies were compared with data from other noninvasive tests as well as surgical follow-up. Results from these examinations indicate that NMR-derived measurements of aortic root diameter agree closely with echocardiographic measurements. In addition, NMR provides more complete anatomic detail than does echocardiography and can be utilized to assess and follow up virtually all patients with this syndrome.

Does indomethacin attenuate the coronary vasodilatory effect of nitroglycerin?

Although previous studies have shown that indomethacin attenuates the dilative effects of nitroglycerin on human peripheral veins and canine coronary arteries, its ability to alter the influence of nitroglycerin on coronary blood flow in human beings is unknown. In 22 patients (16 men and 6 women, aged 47 +/- 10 years [mean +/- standard deviation]) referred for the evaluation of chest pain, heart rate, systemic arterial pressure, coronary sinus blood flow (by thermodilution) and coronary vascular resistance (mean arterial pressure/coronary sinus blood flow) were measured before and during the administration of intracoronary saline solution (n = 6, [control subjects]) or intracoronary nitroglycerin, 100 micrograms (n = 16). Of these 16 patients, 8 had no pretreatment and the other 8 received 50 mg of indomethacin orally, 10 and 2 to 3 hours before study. In the six control subjects, no variable changed with saline injection. In the eight patients given nitroglycerin without indomethacin pretreatment, heart rate and mean systemic arterial pressure were changed modestly (72 +/- 15 to 74 +/- 15 beats/min and 93 +/- 9 to 87 +/- 13 mm Hg, respectively, p less than 0.05), coronary sinus blood flow increased by 56 +/- 43% (107 +/- 72 to 155 +/- 78 ml/min, p less than 0.001) and coronary vascular resistance decreased (1.12 +/- 0.50 to 0.66 +/- 0.26 mm Hg/ml per min, p = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo measurement of myocardial mass using nuclear magnetic resonance imaging.

To examine the accuracy of nuclear magnetic resonance imaging in measuring left ventricular mass, measurements of left ventricular mass made using this technique were compared with left ventricular weight in 10 mongrel dogs. Left ventricular myocardial volume was measured from five short-axis end-diastolic images that spanned the left ventricle. Left ventricular mass was calculated from left ventricular myocardial volume and compared with the left ventricular weight determined after formalin immersion-fixation. Linear regression analysis yielded the following relation in grams: left ventricular mass determined using nuclear magnetic resonance imaging = (0.94) (left ventricular weight) + 9.1 (r = 0.98, SEE = 6.1 g). The small overestimation of left ventricular weight by nuclear magnetic resonance imaging was judged to be secondary to both difficulty with proper border definition and partial volume effects. Hence, this imaging technique can be used to obtain accurate measurements of left ventricular mass in dogs in vivo.

Gadolinium-DTPA-enhanced nuclear magnetic resonance imaging of reperfused myocardium: identification of the myocardial bed at risk.

Gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced nuclear magnetic resonance (NMR) imaging can be useful in the identification of reperfused myocardium. However, the effects of dose and the time of administration and the relation of the extent of the region of enhancement to the myocardial bed at risk have not been evaluated. In this study, dogs were given Gd-DTPA (0.1 mM/kg body weight [n = 21] or 0.34 mM/kg [n = 7]) or saline solution (n = 5) after various periods of occlusion and reperfusion. Twenty-five dogs were killed after 1 or 2 h of reperfusion and the excised hearts were imaged. Images were analyzed for presence, intensity and extent of a region of increased signal. All images in dogs given Gd-DTPA had easily identifiable regions of increased signal in the distribution of the reperfused myocardial bed. Analysis of the extent of these regions in spin-echo images of excised hearts when Gd-DTPA was administered after 5 min of reperfusion demonstrated a correlation coefficient of 0.72 with the bed at risk as determined postmortem with a dye perfusion technique. These images consistently overestimated the infarct size. Signal intensity of the reperfused myocardium increased to a maximum of 1.67 times control (p less than 0.05) in spin-lattice relaxation time (T1)-weighted sequences as the dose of Gd-DTPA increased. This was due to a higher concentration of Gd-DTPA in the reperfused myocardium with resultant shortening of the T1 relaxation time. When Gd-DTPA was given after 90 min of reperfusion, NMR images did not identify the bed at risk.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of metoprolol on myocardial function and energetics in patients with nonischemic dilated cardiomyopathy: a randomized, double-blind, placebo-controlled study.

OBJECTIVES: This study examined the effects of metoprolol on left ventricular performance, efficiency, neurohormonal activation and myocardial respiratory quotient in patients with dilated cardiomyopathy. BACKGROUND: The mechanism by which beta-adrenergic blockade improves ejection fraction in patients with dilated cardiomyopathy remains an enigma. Thus, we undertook an extensive hemodynamic evaluation of this mechanism. In addition, because animal models have shown that catecholamine exposure may increase relative fatty acid utilization, we hypothesized that antagonism of sympathetic stimulation may result in increased carbohydrate utilization. METHODS: This was a randomized, double-blind, prospective trial in which 24 men with nonischemic dilated cardiomyopathy underwent cardiac catheterization before and after 3 months of therapy with metoprolol (n = 15) or placebo (n = 9) in addition to standard therapy. Pressure-volume relations were examined using a micromanometer catheter and digital ventriculography. RESULTS: At baseline, the placebo-treated patients had somewhat more advanced left ventricular dysfunction. Ejection fraction and left ventricular performance improved only in the metoprolol-treated patients. Stroke and minute work increased without an increase in myocardial oxygen consumption, suggesting increased myocardial efficiency. Further increases in ejection fraction were seen between 3 and 6 months in the metoprolol group. The placebo group had a significant increase in ejection fraction only after crossover to metoprolol. A significant relation between the change in coronary sinus norepinephrine and myocardial respiratory quotient was seen, suggesting a possible effect of adrenergic deactivation on substrate utilization. CONCLUSIONS: These data demonstrate that in patients with cardiomyopathy, metoprolol treatment improves myocardial performance and energetics, and favorably alters substrate utilization. Beta-adrenergic blocking agents, such as metoprolol, are hemodynamically and energetically beneficial in the treatment of myocardial failure.

Influence of propranolol on acidosis and high energy phosphates in ischaemic myocardium of the rabbit.

Catecholamines stimulate adenosine triphosphate consumption and glycogenolysis in isolated hearts. In ischaemic myocardium a protective effect of beta adrenergic blockade may therefore arise from reduced adenosine triphosphate consumption or attenuation of acidosis. To characterise the biochemical mechanism of this protective effect phosphorus metabolite concentrations and intracellular pH were measured in an ischaemic region of the rabbit heart in vivo using 31P-nuclear magnetic resonance spectroscopy. After occlusion of the left anterior descending coronary artery there was a rapid decrease in intracellular phosphocreatine concentrations, and intracellular adenosine triphosphate concentrations decreased at a rate of approximately 0.5 mumol X g-1 dry weight X min-1. Intracellular pH decreased approximately linearly to a final pH of about 5.8. In the ischaemic myocardium of the animals treated with propranolol the intracellular concentrations of adenosine triphosphate were higher and those of phosphocreatine lower and the pH was the same compared with control after 30 minutes of ischaemia. Thus any protective effect of propranolol in vivo is not associated with attenuation of intracellular acidosis in this preparation.

Carbon flux through citric acid cycle pathways in perfused heart by 13C NMR spectroscopy.

Mathematical models of the TCA cycle derived previously for 14C tracer studies have been extended to 13C NMR to measure the 13C fractional enrichment of [2-13C]acetyl-CoA entering the cycle and the relative activities of the oxidative versus anaplerotic pathways. The analysis is based upon the steady-state enrichment of 13C into the glutamate carbons. Hearts perfused with [2-13C]acetate show low but significant activity of the anaplerotic pathways. Activation of two different anaplerotic pathways is demonstrated by addition of unlabeled propionate or pyruvate to hearts perfused with [2-13C]acetate. In each case, the amount of [2-13C]acetate being oxidized and the relative carbon flux through anaplerotic versus oxidative pathways are evaluated.

Direct observation of lactate and alanine by proton double quantum spectroscopy in rat hearts supplied with [3-13C]pyruvate.

The 13C-fractional enrichments in the lactate and alanine methyl carbon positions were determined by 1H NMR spectroscopy of extracts of rat hearts perfused with various concentrations of [3-13C]pyruvate +/- unlabeled glucose or acetate. In general, the 13C-fractional enrichment of the alanine methyl carbon pool paralleled the 13C-fractional enrichment of the acetyl-CoA which entered the TCA cycle (as determined by 13C-isotopomer analysis) while the 13C-fractional enrichment of the lactate methyl carbon was always significantly lower, consistent with a pool of lactate which does not mix with exogeneous [3-13C]pyruvate. This has also been examined in intact, perfused, KCl-arrested rat hearts supplied with [3-13C]pyruvate by proton double quantum metabolite specific spectroscopy (MSS). A comparison of MSS spectra of intact hearts with one pulse spectra of extracts of those same hearts indicates there is a sizeable non-enriched pool of lactate in the intact hearts which is not visible by NMR spectroscopy.

NOTPME: a 31P NMR probe for measurement of divalent cations in biological systems.

1,4,7-Triazacyclononane-N,N',N''-tris(methylenephosphonate monoethylester) (NOTPME) has been synthesized, characterized and analyzed for use as a 31P NMR indicator of intracellular Mg2+ and Zn2+ ions. The 31P NMR spectrum of this chelate in the presence of metal ions shows characteristic resonances for the free chelate, Mg(NOTPME)-, Zn(NOTPME)-, and Ca(NOTPME)-. The Kd values indicate that this chelate has a 10-fold higher affinity for Mg2+ than for Ca2+ at physiological pH values. In the presence of Mg2+, NOTPME is readily loaded into red blood cells. A 31P NMR spectrum of red cells taken after several washings shows resonances characteristic of entrapped NOTPME and the Mg(NOTPME)- complex, the relative areas of which report an intracellular free Mg2+ concentration of 0.32 mM. The 31P chemical shifts of the free chelate and its metal complexes are far downfield from the typical phosphorus-containing metabolites observed in biological systems, thus making it possible to monitor intracellular cation concentrations and cell energetics simultaneously.

C isotopomer analysis of glutamate by heteronuclear multiple quantum coherence-total correlation spectroscopy (HMQC-TOCSY).

13C has become an important tracer isotope for studies of intermediary metabolism. Information about relative flux through pathways is encoded by the distribution of 13C isotopomers in an intermediate pool such as glutamate. This information is commonly decoded either by mass spectrometry or by measuring relative multiplet areas in a 13C NMR spectrum. We demonstrate here that groups of glutamate 13C isotopomers may be quantified by indirect detection of protons in a 2D HMQC-TOCSY NMR spectrum and that fitting of these data to a metabolic model provides an identical measure of the 13C fractional enrichment of acetyl-CoA and relative anaplerotic flux to that given by direct 13C NMR analysis. The sensitivity gain provided by HMQC-TOCSY spectroscopy will allow an extension of 13C isotopomer analysis to tissue samples not amenable to direct 13C detection (approximately 10 mg soleus muscle) and to tissue metabolites other than glutamate that are typically present at lower concentrations.

NMR indirect detection of glutamate to measure citric acid cycle flux in the isolated perfused mouse heart.

(13)C-edited proton nuclear magnetic resonance (NMR) spectroscopy was used to follow enrichment of glutamate C3 and C4 with a temporal resolution of approximately 20 s in mouse hearts perfused with (13)C-enriched substrates. A fit of the NMR data to a kinetic model of the tricarboxylic acid (TCA) cycle and related exchange reactions yielded TCA cycle (V(tca)) and exchange (V(x)) fluxes between alpha-ketoglutarate and glutamate. These fluxes were substrate-dependent and decreased in the order acetate (V(tca)=14.1 micromol g(-1) min(-1); V(x)=26.5 micromol g(-1) min(-1))>octanoate (V(tca)=6.0 micromol g(-1) min(-1); V(x)=16.1 micromol g(-1) min(-1))>lactate (V(tca)=4.2 micromol g(-1) min(-1); V(x)=6.3 micromol g(-1) min(-1)).

Effects of different oxidative insults on intermediary metabolism in isolated perfused rat hearts.

13C and 31P NMR were used to evaluate exogenous substrate utilization and endogenous phosphate metabolites in perfused rat hearts exposed to tert-butylhydroperoxide (tert-BOOH) and hydrogen peroxide (H2O2). Both reagents caused a reduction in developed pressure compared to controls and, in agreement with previous 31P NMR data, had different effects on intracellular high-energy phosphates and glycolysis. 13C Isotopomer analysis of tissue extracts showed that H2O2 and tert-BOOH also had significantly different effects on substrate utilization by the citric acid cycle. The contribution of exogenous lactate and glucose to acetyl-CoA was 43% in controls and increased to over 80% in the presence of either oxidant. With tert-BOOH, exogenous glucose and lactate were both significant contributors to acetyl-CoA (44 +/- 2 and 41 +/- 3%). However, with H2O2, exogenous lactate supplied a much higher fraction of acetyl-CoA (72 +/- 2%) than glucose (9 +/- 1%). Also, when [2-(13)C] glucose was supplied, accumulation of [2-(13)C] and [5-(13)C] fructose 1,6-bisphosphate was observed in the presence of H2O2, indicating inhibition of glyceraldehyde-3-phosphate dehydrogenase. These results indicate that despite this glycolytic inhibition, H2O2 increased the utilization of pyruvate precursors when lactate was present as an alternative carbohydrate substrate.

Measurement of gluconeogenesis and pyruvate recycling in the rat liver: a simple analysis of glucose and glutamate isotopomers during metabolism of [1,2,3-(13)C3]propionate.

Simple equations that relate glucose and glutamate 13C-NMR multiplet areas to gluconeogenesis and pyruvate recycling during metabolism of [1,2,3-(13)C3]propionate are presented. In isolated rat livers, gluconeogenic flux was 1.2 times TCA cycle flux and about 40% of the oxaloacetate pool underwent recycling to pyruvate prior to formation of glucose. The 13C spectra of glucose collected from rats after gastric versus intravenous administration of [1,2,3-(13)C3]propionate indicated that pyruvate recycling was slightly higher in vivo (49%) while glucose production was unchanged. This indicates that a direct measure of gluconeogenesis and pyruvate recycling may be obtained from a single 13C-NMR spectrum of blood collected after oral administration of enriched propionate.

Direct determination of the attenuation coefficient for radionuclide volume measurements.

Correcting for the attenuation of photons between the cardiac chambers and chest surface is crucial for accurate nongeometric ventricular volume determinations from equilibrium radionuclide angiograms. Previous techniques have assumed that the attenuation coefficient of water for 99mTc (0.15/cm) should be used for this correction. In this study, this assumption was tested directly by measuring attenuation of the activity of a radioactive source within the right and left cardiac chambers. The balloon of a flow-directed catheter, filled with 99mTc, was used as a source and its depth within the body was measured with biplane fluoroscopy. In ten patients, a total of 36 measurements of attenuation were made. With linear regression analysis, the overall calculated attenuation coefficient, mu, was 0.12/cm (standard error of slope = 0.01, R = 0.93). Although the mean value of mu varied from 0.08 to 0.13 for four different intracardiac locations these differences were not significant. These direct measurements indicate that the attenuation of photons in the heart is not equivalent to that of water and suggest that an attenuation coefficient of 0.12/cm should be used in analyzing ventricular activity.

Improved in vivo magnetic resonance imaging of acute myocardial infarction after intravenous paramagnetic contrast agent administration.

Gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA), a paramagnetic relaxation agent, has been used in vitro to improve the detection of acute myocardial infarction (MI) by magnetic resonance imaging (MRI). In this study, the ability of Gd-DTPA to improve in vivo magnetic resonance imaging of MI was examined. Ten dogs with MI caused by left anterior descending coronary artery ligation were imaged 1 to 5 days after infarction. Imaging was performed before and for 2 hours after intravenous administration of 0.34 mmol/kg Gd-DTPA. One to 2 days after MI, Gd-DTPA improved visualization of the infarct in 3 of 4 dogs. This effect was more prominent in dogs imaged 4 to 5 days after MI, when 6 of 6 dogs showed substantially improved infarct definition after Gd-DTPA. At both times the intensity ratio, an objective measure of contrast between infarcted and normal tissue that is defined by the ratio of image intensity of infarcted area to that of noninfarcted area, was significantly better after Gd-DTPA administration. The intensity ratio at 24 to 48 hours after infarction was 1.4 +/- 0.2 (mean +/- standard deviation) before Gd-DTPA, and 1.7 +/- 0.6 after Gd-DTPA (p = 0.03). The intensity ratio at 4 to 5 days after infarction was 1.5 +/- 0.3 before Gd-DTPA, and 1.8 +/- 0.5 after Gd-DTPA (p less than 0.001). Thus, Gd-DTPA administration improves in vivo visualization of MI by MRI.