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A D Sherry

Publications and source records attributed to A D Sherry.

At least 55 records · Page 3Linked to original sources
PubMed1997

Influence of vanadate on glycolysis, intracellular sodium, and pH in perfused rat hearts.

Vanadium compounds have been shown to cause a variety of biological and metabolic effects including inhibition of certain enzymes, alteration of contractile function, and as an insulin like regulator of glucose metabolism. However, the influence of vanadium on metabolic and ionic changes in hearts remains to be understood. In this study we have examined the influence of vanadate on glucose metabolism and sodium transport in isolated perfused rat hearts. Hearts were perfused with 10 mM glucose and varying vanadate concentrations (0.7-100 microM) while changes in high energy phosphates (ATP and phosphocreatine (PCr)), intracellular pH, and intracellular sodium were monitored using 31P and 23Na NMR spectroscopy. Tissue lactate, glycogen, and (Na+, K+)-ATPase activity were also measured using biochemical assays. Under baseline conditions, vanadate increased tissue glycogen levels two fold and reduced (Na+, K+)-ATPase activity. Significant decreases in ATP and PCr were observed in the presence of vanadate, with little change in intracellular pH. These changes under baseline conditions were less severe when the hearts were perfused with glucose, palmitate and beta-hydroxybutyrate. During ischemia vanadate did not limit the rise in intracellular sodium, but slowed sodium recovery on reperfusion. The presence of vanadate during ischemia resulted in attenuation of acidosis, and reduced lactate accumulation. Reperfusion in the presence of vanadate resulted in a slower ATP recovery, while intracellular pH and PCr recovery was not affected. These results indicate that vanadate alters glucose utilization and (Na+, K+)-ATPase activity and thereby influences the response of the myocardium to an ischemic insult.

3-Hydroxybutyric Acid↗
PubMed1996

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.

Adenine Nucleotides↗
PubMed1996

Isotopic methods for probing organization of cellular metabolism.

These examples serve to illustrate that it is now possible to investigate metabolism in intact tissues using a variety of biophysical methods. While we have concentrated on NMR methods, reflecting our own interests in using 13C as a metabolic tracer, GC-mass spectroscopy can often provide similar metabolic information and has the advantage of increased sensitivity over NMR. Combining either or both of these technologies with cleaver 'chemical biopsy' methods offers new opportunities to examine what may seem to be old metabolic questions in a much more relevant environment, the native state.

Animals↗
PubMed1996

Substrate selection early after reperfusion of ischemic regions in the working rabbit heart.

Several substrates are available in vivo for oxidation by the myocardium. Although substrate selection has been studied extensively in normoxic myocardium, relatively little is known about substrate preference very early during reperfusion after ischemia. Carbon-13 isotopomer analysis was used to study substrate usage by nonischemic and reperfused-ischemic myocardium in a working heart that was subjected to 15 min or regional ischemia and reperfused for 5 min. Compared with nonischemic myocardium, the contribution of acetoacetate to acetyl coenzyme A was increased in the reperfused-ischemic region, and the contribution of exogenous lactate was decreased. Free fatty acid oxidation, however, was not different in the two regions. These results indicate that (1) early during reperfusion, ketone body oxidation may be more significant than has been emphasized, (2) the relative contribution of fatty acids to acetyl coenzyme A is not sensitive to ischemia followed by reperfusion, and (3) Carbon-13 magnetic resonance spectroscopy methods may be used for analysis of spatial heterogeneity of metabolism in the heart.

Acetoacetates↗
PubMed1996

Oxidation of acetate in rabbit skeletal muscle: detection by 13C NMR spectroscopy in vivo.

The results of a proton-decoupled and Overhauser-enhanced 13C NMR study of acetate metabolism in skeletal muscle are reported. [2-13C]Acetate was infused intravenously over 2 h into anesthetized rabbits, and skeletal muscle in the lateral thigh was monitored by 13C NMR spectroscopy at 4.7 T. Stable 13C enrichment in carbons 2, 3, and 4 of glutamate was observed at the end of the infusion, and the half-time for enrichment was 17 min for glutamate C4 and 50 min for glutamate C2 and C3. The contribution of exogenous acetate to acetylcoenzyme A was nearly equal in skeletal muscle and heart in vivo (83-87%, measured in tissue extracts), comparable with earlier perfused heart studies in which acetate was the sole available substrate. Although relative flux through the combined anaplerotic pathways (relative to citric acid cycle flux) was higher in quiescent skeletal muscle (28%) compared with hearts (3%) from the same animals, actual anaplerotic flux was estimated to be substantially higher in heart than in skeletal muscle after correcting for differences in citric acid cycle flux in the two tissues.

Acetates↗
PubMed1996

TmDOTP5-: a substance for NMR temperature measurements in vivo.

The chemical shifts of 31P and 1H in thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) (TmDOTP5-) are approximately two orders of magnitude more sensitive to temperature than are water proton and 19F shifts. In the physiologically relevant pH range, the 31P and 1H chemical shifts of TmDOTP5- are linear functions of temperature between 25 and 47 degrees C. The results indicate that using TmDOTP5- can provide measurements of temperature in vivo that are significantly more accurate than methods based on water and fluorocarbon chemical shifts.

Abdomen↗
PubMed1996

Contribution of various substrates to total citric acid cycle flux and anaplerosis as determined by 13C isotopomer analysis and O2 consumption in the heart.

A simple relationship between parameters derived from a 13C NMR isotopomer analysis and O2 consumption is presented that allows measurement of the absolute rate of acetyl-CoA oxidation and anaplerotic flux in tissues oxidizing a mixture of four substrates. The method was first applied in a study of the effects of work state and beta-adrenergic stimulation on net acetate oxidation and anaplerosis in the isolated working rat heart. The results demonstrate that the anticipated ratio of 2 between O2 consumption and TCA cycle flux for hearts oxidizing only acetate holds at low workload when anaplerosis is low, but deviates toward a factor of 3 under high workload conditions when anaplerosis is increased. This analysis was also extended to hearts that oxidize a more physiological mixture of substrates including long-chain fatty acids, acetoacetate, lactate, pyruvate, and glucose. We show that the contribution each substrate makes to total TCA cycle flux can be determined by combined 13C NMR and O2 consumption measurements. The present study also demonstrates that stimulation of anaplerosis (by addition of propionate) can significantly alter the relative contribution each substrate makes to total TCA cycle flux. We conclude if 13C labeling patterns are selected appropriately, a comprehensive picture of flux through all major metabolic pathways feeding the cycle can be determined in a single experiment even when complex physiological mixtures of substrates are provided.

Acetates↗
PubMed1996

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.

Animals↗
PubMed1996

13C isotopomer model for estimation of anaplerotic substrate oxidation via acetyl-CoA.

A previous model using 13C nuclear magnetic resonance isotopomer analysis provided for direct measurement of the oxidation of 13C-enriched substrates in the tricarboxylic acid cycle and/or their entry via anaplerotic pathways. This model did not allow for recycling of labeled metabolites from tricarboxylic acid cycle intermediates into the acetyl-CoA pool. An extension of this model is now presented that incorporates carbon flow from oxaloacetate or malate to acetyl-CoA. This model was examined using propionate metabolism in the heart, in which previous observations indicated that all of the propionate consumed was oxidized to CO2 and water. Application of the new isotopomer model shows that 2 mM [3-13C]propionate entered the tricarboxylic acid cycle as succinyl-CoA (an anaplerotic pathway) at a rate equal to 52% of tricarboxylic acid cycle turnover and that all of this carbon entered the acetyl-CoA pool and was oxidized. This was verified using standard biochemical analysis; from the rate (mumol.min-1.g dry wt-1) of propionate uptake (4.0 +/- 0.7), the estimated oxygen consumption (24.8 +/- 5) matched that experimentally determined (24.4 +/- 3).

Acetyl Coenzyme A↗
PubMed1996

Effects of ischemia on intracellular sodium and phosphates in the in vivo rat liver.

Metabolic factors that influence the transition form reversible to irreversible ischemic injury were studied in the rat liver in vivo with 31P-nuclear magnetic resonance (NMR) spectroscopy. Hepatic ischemia for 15, 35, or 65 min was produced by occlusion of the hepatic artery and portal vein in rats. Ischemia caused a rapid decrease in the ATP concentration ([ATP])-to-P(i) concentration ratio and pH within 5 min, but there was little change in these variables detectable by 31P-NMR with longer periods of ischemia. After reperfusion, the [ATP] and P(i) concentration returned toward normal values in livers exposed to 15 or 35 min of ischemia, but 65 min of ischemia were associated with only modest recovery in [ATP], and the [ATP] later decreased. Because the 31P-NMR spectrum was similar after brief compared with prolonged ischemia, it appears that neither ATP depletion, P(i) accumulation, nor acidosis predicts metabolic recovery. Hepatic intracellular NA+ was also measured in separate groups of animals by 23Na-NMR in the presence of a shift agent, thulium (III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis (methylene-phosphonate) (TmDOTP5-), and by atomic absorption spectroscopy. Under baseline conditions, the concentration of intracellular Na+ was 15.2 mM by atomic absorption spectroscopy and 16.5 mM by 23Na-NMR. Although the 31P-NMR spectrum responded very rapidly to the onset of ischemia, intracellular Na+ concentration measured by 23Na-NMR increased gradually but steadily at approximately 1.0 mM/min during early (up to 15 min) ischemia. These observations demonstrate that a rise in intracellular Na+ does occur early ischemia, that TmDOTP5- can be applied in vivo for analysis of intracellular Na+ in the ischemic liver, and that 31P-NMR spectroscopy is very sensitive to early ischemic injury.

Adenosine Triphosphate↗
PubMed1995

1H NMR detection of lactate and alanine in perfused rat hearts during global and low pressure ischemia.

A spin-echo method is presented for obtaining high resolution, 13C coupled, proton spectra of lactate and alanine in intact, beating rat hearts. All hearts were depleted of glycogen prior to prolonged perfusion with either 10 mM unenriched glucose or [1-13C]glucose to restore glycogen. These two groups of hearts were then examined by 1H NMR during prolonged global (zero flow) or low pressure (low flow) ischemia. During global ischemia, lactate was derived from both glucose and glycogen, with endogenous glycogen contributing twice as much lactate as exogenous glucose. During low perfusion pressure ischemia, however, lactate was derived exclusively from exogenous glucose. The entire pool of lactate (both 12C and 13C) was visible by NMR in intact, glucose perfused hearts while alanine was not detected. However, upon adding 10 mM pyruvate to the perfusate, the entire alanine pool became NMR visible while some of the lactate became NMR invisible. These observations indicate that the NMR visibility of small, usually highly mobile metabolites such as alanine and lactate is not always 100% in intact hearts and that the NMR visibility of these molecules may depend upon which exogenous substrate is presented to the heart.

Alanine↗
PubMed1995

TmDOTP5- as a 23Na shift reagent for the in vivo rat kidney.

Since transmembrane sodium gradient is essential to many cell functions, there is continuing interest in methods that differentiate intracellular and extracellular Na+. In the kidney, shift reagent (SR) aided 23Na magnetic resonance spectroscopy (MRS) has been successfully used only in isolated cells, tubules, and the perfused organ. In this report, we demonstrate for the first time that TmDOTP5- can be used to distinguish Na+ compartments in kidneys in vivo. Infusion of 80 mM TmDOTP5- without added Ca2+ produced three resolved 23Na resonances, which we have assigned to intracellular Na+, vascular Na+, and intraluminal Na+. In comparison, infusion of 400 mM DyTTHA3- produced two broad and unresolved resonances. The 31P spectra of the cellular high energy phosphate metabolites indicate that TmDOTP5- is safe for in vivo applications. Washout studies suggest that this SR displays renal clearance similar to that of MR imaging contrast agents. However, the glomerular filtration rate (GFR) in animals infused with TmDOTP5- was reduced by 49% compared with the GFR in control animals, perhaps due to the hypotensive effects of the SR. We conclude that TmDOTP5- is effectively cleared from the blood of live animals but that a different formulation will be required for clinical application.

Animals↗
PubMed1995

The design of macrocyclic ligands for monitoring magnesium in tissue by 31P NMR.

A series of ligands based upon 1,4,7-triazacyclononane, containing variable numbers of acetate vs methylenephosphonate ester or methylenephosphonate sidechains, has been synthesized and characterized as possible 31P NMR probes for monitoring [Mg2+]free. The diacetate monophosphonate and diacetate monophosphonate ester mixed ligands proved to have conditional (dissociation) constants at pH 7.4 in an appropriate range for measuring typical levels of intracellular [Mg2+]free, and binding selectivities for Mg2+ over Ca2+ that ranged from 1.4 to 6.8. The 31P resonances of these ligands were well downfield of typical tissue phosphate metabolite resonances, and addition of Mg2+ to any one of these ligands resulted in well-resolved resonances for HL and MgL (where L = ligand), in slow chemical exchange. The chemical shift differences between the HL and MgL species varied from 2.1 to 2.6 ppm for three different ligands. The conditional Mg(2+)-L binding constants were sensitive to pH in the physiological range, due to protonation of a single macrocyclic nitrogen at high pH (log K1 values ranged from 10 to 12). However, given conditions that allow an independent assessment of pH (i.e., from the 31P chemical shift of Pi), we show that accurate KD values can be estimated from the known thermodynamic stability constants (KMgL) and ligand protonation constants (Ki). This makes these ligands potentially useful for monitoring [Mg2+]free by 31P NMR over a variety of solution conditions, even during conditions when the pH may be changing.

Chelating Agents↗
PubMed1995

Preparation, physico-chemical characterization, and relaxometry studies of various gadolinium(III)-DTPA-bis(amide) derivatives as potential magnetic resonance contrast agents.

Macroscopic protonation constants were measured for a series of DTPA mono- and bis-amide ligands using potentiometric titrations. Proton NMR pH titrations yielded protonation populations of the various nitrogen and oxygen basic sites of the ligands for the different protonation stages. Amide formation decreased the basicity of the backbone nitrogens of the ligands and the thermodynamic stability of the corresponding Gd3+ chelates. Nuclear magnetic relaxation dispersion (NMRD) profiles and ESR linewidths were measured for the Gd3+ chelates. Some of these exhibited an elevated high field relaxivity relative to Gd(DTPA)2-, in response to their high molecular weight. As opposed to Gd(DTPA)2-, at 5 degrees C the chemical exchange process of the single inner-sphere water molecule of the bis-amide complexes becomes so slow that it governs the paramagnetic relaxation process, causing the observed NMRD profiles to be close to those expected for the outer-sphere contribution. The chelates containing long alkyl side chains, such as Gd(DTPA-HPA2), showed increased relaxivity values in the presence of human serum albumin (HSA), indicative of noncovalent interaction with the protein. These chelates could be useful as nonionic hepatobiliary contrast agents.

Contrast Media↗
PubMed1995

Direct evidence that perhexiline modifies myocardial substrate utilization from fatty acids to lactate.

Perhexiline maleate, originally classified as a calcium antagonist, is in use as an antianginal agent. The mechanism of its protective effect is unknown, but there is speculation that it involves a modification of myocardial substrate utilization, in which glycolytic sources are used rather than fatty acids. This hypothesis was tested by employing [13C]NMR isotopomer analysis to measure substrate selection in the working rat heart. Substrate utilization was measured from a mixture of substrates present at their physiological concentration, as follows: acetoacetate, glucose, lactate and long-chain fatty acids. Control perfusions were compared with those perfused with perhexiline. It was found that perhexiline increased lactate utilization, which reduced the extent of fatty acid and endogenous substrate oxidation. There was also a significant increase in cardiac output for a small and insignificant increase in oxygen consumption, which suggested an improvement in myocardial efficiency. Thus, it was confirmed by direct measurement that this drug does modify substrate oxidation, which suggests that further investigations of the role that this agent can play in the management of ischemic heart disease would be beneficial.

Acetoacetates↗
PubMed1994

A 13C isotopomer n.m.r. method for monitoring incomplete beta-oxidation of fatty acids in intact tissue.

An n.m.r. method is presented for monitoring the extent to which fatty acids undergo beta-oxidation without release of shorter-chain intermediates. It is based upon a 13C isotopomer analysis of glutamate from tissue presented with a mixture of [2,4,6,8-13C]octanoate and [1,2,3,4-13C]octanoate. The method does not require steady-state metabolic or isotopic conditions, so it may be applied during a variety of metabolic circumstances, including perfused tissue under stress and in vivo. We have tested the method in perfused rat hearts during anoxia, a model where previous work has shown that beta-oxidation of palmitate is incomplete and shorter-chain intermediates are released [Rabinowitz and Hercker (1974) Arch. Biochem. Biophys. 161, 621-627]. Indeed, n.m.r. spectra of freeze-clamped, acid-extracted tissue show that octanoate undergoes complete beta-oxidation in control normoxic rat hearts, but not in anoxic hearts. Complete beta-oxidation of octanoate was observed under a number of other metabolic conditions in perfused rat hearts, including low-pressure-induced ischaemia, KCl arrest and in the presence of high concentrations of competing substrates. We also demonstrate that the technique is applicable in intact tissue by taking direct measurements in perfused rat hearts using a recently published [13C]homonuclear decoupling technique and in in vivo heart and liver removed from rats after an intravenous infusion of a mixture of [2,4,6,8-13C]octanoate and [1,2,3,4-13C]octanoate.

Animals↗
PubMed1994

Orientation-conserved transfer of symmetric Krebs cycle intermediates in mammalian tissue.

Metabolism of [2-13C]-, [3-13C]-, and [1,2,3-13C]propionate in perfused rat livers and [2-13C]-acetate in perfused rat hearts has been examined in tissue extracts by 13C NMR. Label from [2-13C]-propionate was preferentially incorporated into the C2 carbon of lactate, alanine, and aspartate in liver tissue while label from [3-13C]propionate appeared preferentially in the C3 carbon of those same molecules. These data suggest that 13C may not be completely randomized in the symmetric citric acid cycle intermediates succinate and fumarate as is normally assumed but that some fraction of those intermediates may be transferred between enzymes in this span of the cycle with conservation of spatial orientation, consistent with recent results obtained in yeast [Sumegi et al. (1990) Biochemistry 29, 9106-9110]. This was confirmed by performing similar experiments with [1,2,3-13C]propionate. Time-dependent asymmetry was also observed between the intensities of the glutamate C2 and C3 resonances and between the aspartate C2 and C3 resonances in 13C NMR spectra of intact hearts and heart extracts during early perfusion with [2-13C]-acetate. A model is presented which predicts that isotopic asymmetry is observed only during the first 2-3 turns of the cycle pools when isotope enters the cycle via acetyl-CoA even if all symmetric cycle intermediates retain a unique molecular orientation on each pass through the citric acid cycle.

Acetates↗