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).

Alterations in substrate utilization in the reperfused myocardium: a direct analysis by 13C NMR.

An alternative 13C NMR method which allows direct determination of substrate oxidation in tissue for up to three competing 13C-enriched substrates is presented. Oxidation of competing substrates can be measured by 13C NMR spectroscopy under non-steady-state conditions if the relative areas of the glutamate C3 and C4 resonances can be determined. The accuracy of this measurement is limited during brief exposure to 13C-enriched substrates because of the low enrichment in the C3 carbon.

Influence of cardiac pacing on intracellular sodium in the isolated perfused rat heart.

Increased myocardial contractile tension has been described as being associated with increased heart rate. This phenomenon is believed due to greater sodium influx than efflux, resulting in accumulation of intracellular sodium. Sodium-23 nuclear magnetic resonance spectroscopy in combination with extracellular shift reagents offers near-continuous measurements of intracellular sodium that may be correlated with mechanical performance.

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.

Hepatobiliary delivery of polyaminopolycarboxylate chelates: synthesis and characterization of a cholic acid conjugate of EDTA and biodistribution and imaging studies with its indium-111 chelate.

A conjugate in which the steroid nucleus of cholic acid was linked to EDTA via an 11-atom spacer was obtained by reacting the succinimidyl ester of cholic acid with the amine formed by reaction of a benzyl isothiocyanate derivative of EDTA with N-(tert-butoxycarbonyl)ethylenediamine and subsequent deprotection. Potentiometric titration studies with model complexes showed that the EDTA moiety retained the ability to form 1:1 chelates of high thermodynamic stability, although formation constants were some 3-4 log K units lower for complexes of the conjugate than for the analogous chelates with underivatized EDTA. A complex formed between the cholic acid-EDTA conjugate and 111InIII was clearly rapidly into the liver when injected iv into mice, with subsequent excretion from the liver into the gastrointestinal tract being complete within 1 h of injection.

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.

Channeling of TCA cycle intermediates in cultured Saccharomyces cerevisiae.

Oxidation of [3-13C]propionate was studied in cultured yeast cells, and the distribution of label in the 2- and 3-positions of alanine was detected by 13C NMR. [3-13C]Propionate forms [2-13C]succinyl-CoA in the mitochondria which then enters the citric acid cycle and forms malate through two symmetrical intermediates, succinate and fumarate. If these symmetrical intermediates randomly diffuse from one enzyme to the next in mitochondria as is normally assumed, then 13C labeling in malate C2 and C3 must be equal.

Analysis of tricarboxylic acid cycle of the heart using 13C isotope isomers.

13C-nuclear magnetic resonance (NMR) spectroscopy provides a new approach to the analysis of metabolic pathways, because it detects an interaction between adjacent 13C nuclei. Previous models of isotope distribution in the tricarboxylic acid cycle were designed for analysis of radioisotope data and did not consider the information provided by 13C-13C coupling. A mathematical model of the tricarboxylic acid cycle was developed that preserves all isotope isomer (isotopomer) information and yields simple relationships between 13C-NMR spectra of glutamate and metabolic parameters under steady-state conditions.

Contribution of exogenous substrates to acetyl coenzyme A: measurement by 13C NMR under non-steady-state conditions.

A method is presented for the rapid determination of substrate selection in a manner that is not restricted to conditions of metabolic and isotopic steady state. Competition between several substrates can be assessed directly and continuously in a single experiment, allowing the effect of interventions to be studied. It is shown that a single proton-decoupled 13C NMR spectrum of glutamate provides a direct measure of the contribution of exogenous 13C-labeled substrates to acetyl-CoA without measurement of oxygen consumption and that steady-state conditions need not apply.

Influence of global ischemia on intracellular sodium in the perfused rat heart.

Intracellular [Na+], [H+], and [ATP] and mechanical performance were measured in the isovolumic perfused rat heart during ischemia. The concentration of intracellular sodium, [Na+]i, was determined by atomic absorption spectroscopy under control conditions, and [Na+]i was monitored by 23Na NMR spectroscopy at 1-min intervals under control conditions and during global ischemia. [ATP], [H+], and [Pi] were measured by 31P NMR in a separate group under identical conditions.

Tm(DOTP)5-: a 23Na+ shift agent for perfused rat hearts.

Tm(DOTP)5-, the thulium(III) complex of 1,4,7,10-tetraazacyclododecane-N,N',N',N'''-tetra(methylenephos p hon ate), is introduced as a 23Na+ shift agent for use in discrimination of the NMR resonances of intra- and extracellular 23Na+ ions in perfused rat hearts. The novel shift agent is directly compared to the widely used Dy(TTHA)3- (dysprosium(III) triethylenetetraminehexaacetate).

13C NMR of methylated lysines of fd gene 5 protein: evidence for a conformational change involving lysine 24 upon binding of a negatively charged lanthanide chelate.

Helical complexes formed between fd DNA and reductively methylated fd gene 5 protein were indistinguishable by electron microscopy from complexes formed with the nonmethylated protein. 13C NMR spectroscopy of 13C-enriched N epsilon, N epsilon-dimethyllsyl residues of the protein showed that three of these residues (Lys-24, Lys-46, and Lys-69) were selectively perturbed by binding of the oligomer d(pA)7. These were the same lysyl residues that we previously found to be most protected from methylation by binding of the protein to poly[r(U)] [Dick, L.

Evaluation of polyaza macrocyclic methylene phosphonate chelates of Gd3+ ions as MRI contrast agents.

Gd(DTPA)2- (diethylenetriaminepentaacetic acid) and the polyaza macrocyclic Gd(DOTA)- (1,4,7,10-tetraazacyclododecane-N,N',N'',N''') are paradigms of general purpose paramagnetic complexes useful for enhancing contrast in magnetic resonance imaging (MRI). It is of both fundamental and practical interest to determine how one might modify the chemical structure of these chelate complexes to improve their utility for MRI in specific circumstances. In the present work, we investigated polyaza methylene phosphonate complexes of Gd3+ ions to compare their NMRD profiles with those of their carboxylate analogs and with Gd(DTPA)2-.

Reductive methylation and 13C NMR studies of the lysyl residues of fd gene 5 protein. Lysines 24, 46, and 69 may be involved in nucleic acid binding.

We have examined the role of lysyl residues in the binding of fd gene 5 protein to a nucleic acid polymer. The lysyl residues of the protein were chemically modified to form N epsilon, N epsilon-dimethyllysyl derivatives containing 13C-enriched methyl groups. The 13C NMR spectrum of the modified protein was studied as a function of pH and salt concentration.

Number of inner-sphere water molecules in Gd3+ and Eu3+ complexes of DTPA-amide and -ester conjugates.

The inner-sphere water coordination number for Eu3+ and Gd3+ complexed with five DTPA analogs, in which one or two terminal carboxylate groups are functionalized as propyl amides or propyl esters, have been studied using phosphorescence lifetime and nuclear magnetic relaxation dispersion (NMRD) measurements. Both methods show that the water coordination number does not increase above that observed for the analogous DTPA complexes. The phosphorescence lifetime results indicate that all five Eu3+ complexes have one inner-sphere water molecule at 25 degrees C.

Stability constants for Gd3+ binding to model DTPA-conjugates and DTPA-proteins: implications for their use as magnetic resonance contrast agents.

Five DTPA-amide and ester derivatives have been synthesized and their Gd3+ stability constants have been measured using a simple spectrophotometric method. These results are compared to stability constants measured for Gd3+ binding to two different DTPA-conjugated proteins. Although the thermodynamic constants for Gd3+ binding to DTPA-monopropylamide and DTPA-monopropylester relative to Gd(DTPA)2- decrease by log K = 2.

Propionate metabolism in the rat heart by 13C n.m.r. spectroscopy.

High-resolution 13C n.m.r.

Evaluation of carbon flux and substrate selection through alternate pathways involving the citric acid cycle of the heart by 13C NMR spectroscopy.

A previous 13C NMR technique (Malloy, C. R., Sherry, A.

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.

Organizational aspects of the citric acid cycle.

The enzymes of the citric acid cycle show at least two levels of organization within the mitochondrial matrix. Six of the possible eight sequential enzymes show specific interactions in vitro. Further, the enzymes bind specifically to the matrix surface of the inner membrane.

Magnetic field dependence of solvent proton relaxation rates induced by Gd3+ and Mn2+ complexes of various polyaza macrocyclic ligands: implications for NMR imaging.

The magnetic field dependence of the solvent water proton longitudinal relaxation rate 1/T1 (the NMRD profile) has been measured for solutions of chelates of Gd3+ and Mn2+ ions with two different polyaza macrocyclic ligands: 1,4,7-triazacyclononane-N,N',N",-triacetic acid (NOTA) and 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA). Studies were carried out mainly near physiological pH, but the pH dependence was also examined in some cases. The results are compared with published data for complexes of Gd3+ and Mn2+ ions with ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA).

Metabolic studies of pyruvate- and lactate-perfused guinea pig hearts by 13C NMR. Determination of substrate preference by glutamate isotopomer distribution.

Guinea pig hearts perfused in vitro with 99% enriched [3-13C]pyruvate and [3-13C]lactate have been examined by 13C NMR spectroscopy at 75.45 MHz. Resonances from the intracellular metabolites, glutamate, aspartate, alanine, citrate, malate, lactate, and acetylcarnitine are detected in the [3-13C]pyruvate-perfused hearts while glutamate is the only metabolite observed in the [3-13C]lactate-perfused hearts.

Reductive methylation of the lysyl residues in the fd gene 5 DNA-binding protein: CD and 13C NMR results on the modified protein.

The epsilon-amino groups of the six lysyl residues of the fd gene 5 DNA-binding protein have been modified by reductive methylation to form N epsilon, N epsilon-dimethyl lysyl derivatives containing 13C-labeled methyl groups. The alpha-amino terminus of the protein was not accessible to methylation. Circular dichroism studies show that the modified protein binds to fd DNA, but with a slightly reduced affinity compared with that of unmodified gene 5 protein.