Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Inibidores da Aromatase , Neoplasias da Mama , Receptor ErbB-2 , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Inibidores da Aromatase/uso terapêutico , Feminino , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Receptor ErbB-2/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Aminopiridinas , BenzimidazóisRESUMO
BACKGROUND: Contrast agents (CA) are administered in magnetic resonance imaging (MRI) clinical exams to measure tissue perfusion, enhance image contrast between adjacent tissues, or provide additional biochemical information in molecular MRI. The efficacy of a CA is determined by the tissue distribution of the agent and its concentration in the extracellular space of all tissues. METHODS: In this work, micro-synchrotron radiation x-ray fluorescence (µ-SRXRF) was used to examine and characterize a gadolinium-based zinc-sensitive agent (GdL2) currently under development for detection of prostate cancer (PCa) by MRI. Prostate tissue samples were collected from control mice and mice with known PCa after an MRI exam that included injection of GdL2. The samples were raster scanned to investigate trends in Zn, Gd, Cu, Fe, S, P, and Ca. RESULTS: Significant Zn and Gd co-localization was observed in both healthy and malignant tissues. In addition, a marked decrease in Zn was found in the lateral lobe of the prostate obtained from mice with PCa. CONCLUSION: We demonstrate here that µ-SRXRF is a useful tool for monitoring the distribution of several elements including Zn and Gd in animal models of cancer. The optimized procedures for tissue preparation, processing, data collection, and analysis are described.
Assuntos
Neoplasias da Próstata , Oligoelementos , Animais , Meios de Contraste , Fluorescência , Gadolínio/análise , Humanos , Imageamento por Ressonância Magnética , Masculino , Camundongos , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Espectrometria por Raios X , Síncrotrons , Oligoelementos/análise , Raios X , Zinco/análiseRESUMO
This study was designed to determine the effects of deuteration in pyruvate on exchange reactions in alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and flux through pyruvate dehydrogenase (PDH). Although deuteration of a 13C enriched substrate is commonly used to increase the lifetime of a probe for hyperpolarization experiments, the potential impact of kinetic isotope effects on such substitutions has not been studied in detail. Metabolism of deuterated pyruvate was investigated in isolated rat hearts. Hearts were perfused with a 1:1 mixture of [U-13C3]pyruvate and [2-13C1]pyruvate or a 1:1 mixture of [U-13C3]pyruvate plus [2-13C1, U-2H3]pyruvate for 30â¯min before being freeze clamped. Another set of hearts received [2-13C1, U-2H3]pyruvate and was freeze-clamped at 3â¯min or 6â¯min. Tissue extracts were analyzed by 1H and 13C{1H} NMR spectroscopy. The chemical shift isotope effect of 2H was monitored in the 13C NMR spectra of the C2 resonance of lactate and alanine plus the C5 of glutamate. There was little kinetic isotope effect of 2H in pyruvate on flux through PDH, LDH or ALT as detected by the distribution of 13C, but the distribution of 2H differed markedly between alanine and lactate. At steady-state, alanine was a mixture of deuterated species, while lactate was largely perdeuterated. Consistent with results at steady-state, hearts freeze-clamped at 3â¯min or 6â¯min showed rapid removal of deuterium in alanine but not in lactate. Metabolism of hyperpolarized [1-13C1]pyruvate was compared to [1-13C1,U-2H3]pyruvate in isolated hearts. Consistent with the results from tissue extracts, there was little effect of deuteration on the kinetics of appearance of lactate, alanine or bicarbonate, but there was a small, time-dependent upfield chemical shift in the HP[1-13C1]alanine signal reflecting exchange of methyl deuterons with water protons. Together, these results demonstrate that (1) the kinetics of pyruvate metabolism in hearts detected by 13C NMR are not affected by replacement of the pyruvate methyl protons with deuterons and (2) that the loss of deuterium from the methyl position occurs rapidly during the conversion of pyruvate to alanine. The majority of the deuterium atoms are lost on the time-scale of a hyperpolarization experiment.
Assuntos
Deutério/química , Miocárdio/metabolismo , Ácido Pirúvico/metabolismo , Alanina/metabolismo , Alanina Transaminase/metabolismo , Aminação , Animais , Isótopos de Carbono , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Espectroscopia de Ressonância Magnética , Oxirredução , Complexo Piruvato Desidrogenase/metabolismo , Ratos , Ratos Sprague-Dawley , Água/química , Água/metabolismoRESUMO
Dy complexes can act as suitable negative (T2) contrast agents for Magnetic Resonance Imaging (MRI). As clinical MRI moves toward higher fields, tuning of the exchange rate of coordinated water molecules will become necessary to optimize the r2 relaxivity. For Dy complexes, this will require lengthening of the water residence time, a strategy opposite that required to optimize the r1 relaxivity of Gd complexes. However, very slow water exchange can be deleterious. This is illustrated here by a Dy complex that is characterized by a very slow water exchange. This complex, Dy-DOTA-4AmCE, is compared with several Dy-DTPA derivatives known for their efficacy as T2 contrast agents at high magnetic fields.
Assuntos
Meios de Contraste/química , Imageamento por Ressonância Magnética/métodos , Compostos Organometálicos/química , Ácido Pentético/análogos & derivados , Ácido Pentético/química , Água Corporal , Meios de Contraste/síntese química , Disprósio , Humanos , LigantesRESUMO
Three derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonic acid) (DOTP) containing a hydrophobic substituent on one side chain were prepared and their lanthanide complexes examined by NMR. The new ligands include 1-(1-octyl-methyl-phosphonic acid)-4,7,10-tris(methylene phosphonic acid)-1,4,7,10-tetraazacyclododecane (C(8)-DOTP), 1-(1-undecyl-methyl-phosphonic acid)-4,7,10-tris(methylene phosphonic acid)-1,4,7,10-tetraazacyclododecane (C(11)-DOTP), and 1-(1-4-nitro-phenyl-methyl-phosphonic acid)-4,7,10-tris(methylene phosphonic acid)-1,4,7,10-tetraazacyclododecane (NO(2)-Ph-DOTP). (1)H NMR spectra of the ytterbium(III) complexes were assigned by using a combination of COSY spectroscopy and a fitting procedure that matches experimental NMR hyperfine shifts with those estimated from a MMX-derived structure. The analysis showed that a single isomer is present in solution and that the bulky hydrophobic substituent occupies the less sterically demanding H(6) equatorial position in the YbL(5)(-) complexes. Although the YbL(5)(-) complexes have lower symmetry due to the added substituent, the average (1)H hyperfine shifts are 5-10% larger in these complexes compared to YbDOTP(5)(-). This was magnified further in the hyperfine (23)Na NMR shifts of ion-paired sodium ions where the extracellular Na(+) signal in perfused rat hearts displayed a 28% larger hyperfine shift in the presence of Tm(C(11)-DOTP)(5)(-) than with an equivalent amount of TmDOTP(5)(-).
Assuntos
Elementos da Série dos Lantanídeos/química , Elementos da Série dos Lantanídeos/síntese química , Organofosfonatos/síntese química , Compostos Organofosforados/química , Animais , Fenômenos Químicos , Físico-Química , Coração/efeitos dos fármacos , Hidrólise , Técnicas In Vitro , Ligantes , Espectroscopia de Ressonância Magnética , Masculino , Conformação Molecular , Estrutura Molecular , Nitrobenzenos , Organofosfonatos/química , Ratos , Ratos Sprague-Dawley , Sódio/química , Estereoisomerismo , Túlio/química , Itérbio/químicaRESUMO
The 20 MHz water relaxivity (r(1)) of gadolinium(III) complexes formed with two fatty acid analogues of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) were shown to increase substantially in the presence of albumin. The r(1) values of Gd(C(8)-DOTP)(5-) and Gd(C(11)-DOTP)(5-) in water were similar to that of the parent GdDOTP(5-), a q = 0 complex known to relax water very efficiently via an outer-sphere mechanism. Neither fatty acid analogue formed apparent aggregates or micelles in water up to 20 mM, but both showed dramatic increases in r(1) upon addition of albumin. Further ultrafiltration studies of Gd(C(11)-DOTP)(5-) in the presence of non-defatted HSA showed that the complex binds at a minimum of five high-affinity fatty acid sites with stepwise binding constants ranging from 1.27 x 10(5) to 2.7 x 10(3) M(-1). The 20 MHz relaxivity of Gd(C(11)-DOTP)(5-) in the presence of excess HSA was 23 mM(-1) s(-1) at 25 degrees C. The NMRD curve showed a broad maximum 20-30 MHz which fitted well to standard theory for a q = 0 complex with rapid outer-sphere water exchange. The r(1b) of Gd(C(11)-DOTP)(5-) bound at the tightest site on HSA was approximately 40 mM(-1) s(-1) at 5 degrees C, an extraordinarily high value for an outer-sphere complex. However, the r(1b) of Gd(C(11)-DOTP)(5-) bound at the weaker sites on HSA was considerably lower, approaching the relaxivity of the free complex in water. This suggests that the complex bound in the highest affinity fatty acid site is less mobile than the same complex bound at the weaker affinity fatty acid sites. This combined ultrafiltration and relaxivity study demonstrates that the common assumption of a single r(1b) value for a Gd(3+) complex bound at several protein sites is not a valid approximation.
Assuntos
Ácidos Graxos/química , Gadolínio/química , Compostos Organometálicos/síntese química , Compostos Organofosforados/química , Sarcosina/análogos & derivados , Algoritmos , Sítios de Ligação , Fenômenos Químicos , Físico-Química , Compostos de Dansil/química , Ácidos Graxos/metabolismo , Fluorescência , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Compostos Organometálicos/química , Organofosfonatos/síntese química , Organofosfonatos/química , Proteínas/química , Sarcosina/química , Albumina Sérica/química , Albumina Sérica/metabolismo , Água/química , Água/fisiologiaRESUMO
(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)).
Assuntos
Ciclo do Ácido Cítrico/fisiologia , Ácido Glutâmico/análise , Espectroscopia de Ressonância Magnética/métodos , Miocárdio/metabolismo , Animais , Feminino , Ácido Glutâmico/metabolismo , Técnicas In Vitro , Ácidos Cetoglutáricos/metabolismo , Camundongos , Consumo de Oxigênio , Perfusão , Sensibilidade e EspecificidadeRESUMO
Hepatic glucose synthesis from glycogen, glycerol, and the tricarboxylic acid (TCA) cycle was measured in five overnight-fasted subjects by (1)H, (2)H, and (13)C NMR analysis of blood glucose, urinary acetaminophen glucuronide, and urinary phenylacetylglutamine after administration of [1,6-(13)C(2)]glucose, (2)H(2)O, and [U-(13)C(3)]propionate. This combination of tracers allows three separate elements of hepatic glucose production (GP) to be probed simultaneously in a single study: 1) endogenous GP, 2) the contribution of glycogen, phosphoenolpyruvate (PEP), and glycerol to GP, and 3) flux through PEP carboxykinase, pyruvate recycling, and the TCA cycle. Isotope-dilution measurements of [1,6-(13)C(2)] glucose by (1)H and (13)C NMR indicated that GP in 16-h-fasted humans was 10.7 +/- 0.9 micromol.kg(-1).min(-1). (2)H NMR spectra of monoacetone glucose (derived from plasma glucose) provided the relative (2)H enrichment at glucose H-2, H-5, and H-6S, which, in turn, reflects the contribution of glycogen, PEP, and glycerol to total GP (5.5 +/- 0.7, 4.8 +/- 1.0, and 0.4 +/- 0.3 micromol.kg(-1).min(-1), respectively). Interestingly, (13)C NMR isotopomer analysis of phenylacetylglutamine and acetaminophen glucuronide reported different values for PEP carboxykinase flux (68.8 +/- 9.8 vs. 37.5 +/- 7.9 micromol.kg(-1).min(-1)), PEP recycling flux (59.1 +/- 9.8 vs. 27.8 +/- 6.8 micromol.kg(-1).min(-1)), and TCA cycle flux (10.9 +/- 1.4 vs. 5.4 +/- 1.4 micromol.kg(-1).min(-1)). These differences may reflect zonation of propionate metabolism in the liver.
Assuntos
Acetaminofen/análogos & derivados , Glicemia/metabolismo , Ciclo do Ácido Cítrico , Gluconeogênese , Glucose/metabolismo , Glutamina/análogos & derivados , Fígado/metabolismo , Acetaminofen/urina , Adulto , Isótopos de Carbono , Deutério , Feminino , Glutamina/urina , Humanos , Hidrogênio , Cinética , Glicogênio Hepático/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Modelos Biológicos , Fosfoenolpiruvato/metabolismo , Propionatos/metabolismo , Valores de Referência , Fatores de Tempo , Água/metabolismoRESUMO
Lanthanide complexes of a tetra-amide derivative of DOTA (structure 4 in text) with four extended carboxymethyl esters have been characterized by X-ray crystallography and multinuclear NMR spectroscopy. [Eu(4)(H(2)O)](triflate)(3) crystallized from water in the monoclinic, P(21/)(c) space group (a = 10.366 A, b = 22.504 A, c = 23.975 A, and beta = 97.05 degrees ). The Eu(3+) cation is bound to four macrocyclic nitrogen atoms (mean Eu-N = 2.627 A) and four amide oxygen atoms (mean Eu-O(amide) = 2.335 A) in a square antiprismatic geometry with a twist angle of 38.5 degrees between the N4 and O4 planes. A single bound water molecule (Eu-O(W) = 2.414 A) occupies a typical monocapped position on the O4 surface. In pure water, resonances corresponding to a single Eu(3+)-bound water molecule were observed in the (1)H (53 ppm) and (17)O (-897 ppm) NMR spectra of [Eu(4)(H(2)O)](triflate)(3) at 25 degrees C. A fit of the temperature-dependent Eu(3+)-bound (1)H and (17)O water resonance line widths in acetonitrile-d(3) (containing 4% v/v (17)O enriched water) gave identical lifetimes (tau(m)(298)) of 789 +/- 50 micros (in water as solvent; a line shape analysis of the Eu(3+)-bound water resonance gave a tau(m)(298) = 382 +/- 5 micros). Slow water exchange was also evidenced by the water proton relaxivity of Gd(4) (R(1) = 2.2 mM(-1) s(-1), a value characteristic of pure outer-sphere relaxation at 25 degrees C). With increasing temperature, the inner-sphere contribution gradually increased due to accelerated chemical exchange between bound water and bulk water protons. A fitting of the relaxation data (T(1)) to standard SBM theory gave a water proton lifetime (tau(m)(298)) of 159 micros, somewhat shorter than the value determined by high-resolution (1)H and (17)O NMR of Eu(4). Exchange of the bound water protons in Gd(4) with bulk water protons was catalyzed by addition of exogenous phosphate at 25 degrees C (R(1) increased to 10.0 mM(-1) s(-1) in the presence of 1500-fold excess HPO(4)(2-)).
Assuntos
Metais Terras Raras/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Isótopos de Oxigênio , Prótons , Solventes/química , Temperatura , Água/químicaRESUMO
This study was designed to test the hypothesis that indirect (1)H[(13)C] detection of tricarboxylic acid (TCA) cycle intermediates using heteronuclear multiple quantum correlation-total correlation spectroscopy (HMQC-TOCSY) nuclear magnetic resonance (NMR) spectroscopy provides additional (13)C isotopomer information that better describes the kinetic exchanges that occur between intracellular compartments than direct (13)C NMR detection. NMR data were collected on extracts of rat hearts perfused at various times with combinations of [2-(13)C]acetate, propionate, the transaminase inhibitor aminooxyacetate, and (13)C multiplet areas derived from spectra of tissue glutamate were fit to a standard kinetic model of the TCA cycle. Although the two NMR methods detect different populations of (13)C isotopomers, similar values were found for TCA cycle and exchange fluxes by analyzing the two data sets. Perfusion of hearts with unlabeled propionate in addition to [2-(13)C]acetate resulted in an increase in the pool size of all four-carbon TCA cycle intermediates. This allowed the addition of isotopomer data from aspartate and malate in addition to the more abundant glutamate. This study illustrates that metabolic inhibitors can provide new insights into metabolic transport processes in intact tissues.
Assuntos
Ciclo do Ácido Cítrico/fisiologia , Espectroscopia de Ressonância Magnética , Miocárdio/metabolismo , Ácido Acético/metabolismo , Ácido Acético/farmacocinética , Ácido Amino-Oxiacético/farmacologia , Animais , Ácido Aspártico/metabolismo , Isótopos de Carbono/análise , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico/metabolismo , Coração/efeitos dos fármacos , Técnicas In Vitro , Cinética , Espectroscopia de Ressonância Magnética/métodos , Malatos/metabolismo , Modelos Biológicos , Perfusão , Propionatos/metabolismo , Propionatos/farmacocinética , Ratos , Transaminases/antagonistas & inibidoresRESUMO
The lanthanide complex, thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (TmDOTA-), has been investigated as an agent for MR thermometry in vivo. The chemical shifts of the TmDOTA- protons were highly sensitive to temperature at a clinically relevant field strength, yet insensitive to pH and the presence of Ca2+. Given the excellent stability of lanthanide-DOTA complexes and high thermal sensitivity, TmDOTA- is expected to be a good candidate for MR thermometry in vivo.
Assuntos
Quelantes , Compostos Heterocíclicos com 1 Anel , Espectroscopia de Ressonância Magnética , Animais , Cálcio/química , Quelantes/química , Compostos Heterocíclicos com 1 Anel/química , Concentração de Íons de Hidrogênio , Modelos Anatômicos , Prótons , Ratos , TemperaturaRESUMO
13C NMR isotopomer analysis is a powerful method for measuring metabolic fluxes through pathways intersecting in the tricarboxylic acid cycle. However, the inherent insensitivity of 13C NMR spectroscopy makes application of isotopomer analysis to small tissue samples (mouse tissue, human biopsies, or cells grown in tissue culture) problematic. (1)H NMR is intrinsically more sensitive than 13C NMR and can potentially supply the same information via indirect detection of 13C providing that isotopomer information can be preserved. We report here the use of J-resolved HSQC (J-HSQC) for 13C isotopomer analysis of tissue samples. We show that J-HSQC reports isotopomer multiplet patterns identical to those reported by direct 13C detection but with improved sensitivity.
Assuntos
Carbono/química , Técnicas de Química Analítica/métodos , Hidrogênio/química , Espectroscopia de Ressonância Magnética/métodos , Perfusão , Animais , Ácido Glutâmico/análise , Camundongos , Reperfusão Miocárdica , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Two derivatives of 1,4,7,10-tetraazacyclododecane with trans-acetate and trans-amide side-chain ligating groups have been prepared and their complexes with lanthanide cations examined by multinuclear NMR spectroscopy. These lanthanide complexes exist in aqueous solution as a mixture of slowly interconverting coordination isomers with 1H chemical shifts similar to those reported previously for the major (M) and minor (m) forms of the tetraacetate ([Ln(dota)]-) and tetraamide ([Ln(dtma)]3+) complexes. As in the [Ln(dota)]- and [Ln(dtma)]3+ complexes, the m/M ratio proved to be a sensitive function of lanthanide size and temperature. An analysis of 1H hyperfine shifts in spectra of the Yb3+ complexes revealed significant differences between the axial (D1) and non-axial (D2) components of the magnetic susceptibility tensor anisotropy in the m and M coordination isomers and the energetics of ring inversion and m <==> M isomerization as determined by two-dimensional exchange spectroscopy (EXSY). (17)O shift data for the Dy3+ complexes showed that both have one inner-sphere water molecule. A temperature-dependent (17)O NMR study of bulk water linewidths for solutions of the Gd3+ complexes provided direct evidence for differences in water exchange rates for the two coordination isomers. The bound-water lifetimes (tauM298) in the M and m isomers of the Gd3+ complexes ranged from 1.4-2.4 micros and 3-14 ns, respectively. This indicates that 1) the inner-sphere water lifetimes for the complexes with a single positive charge reported here are considerably shorter for both coordination isomers than the corresponding values for the [Gd(dtma)]3+ complex with three positive charges, and 2) the difference in water lifetimes for M and m isomers in these two series is magnified in the [Gd[dota-bis(amide)]] complexes. This feature highlights the remarkable role of both charge and molecular geometry in determining the exchange rate of the coordinated water.
Assuntos
Quelantes/química , Compostos Heterocíclicos com 1 Anel/química , Metais Terras Raras/química , Água/química , Isomerismo , Cinética , Espectroscopia de Ressonância Magnética , TemperaturaRESUMO
RATIONALE AND OBJECTIVES: Slow water exchange in Gd3+ complexes is generally considered detrimental to their use as MR contrast agents. The objective of this work was to demonstrate how this feature may serve as a useful template for the design of responsive MR agents. METHODS: Lanthanide (Ln) complexes of two 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA)-tetraamide phosphonate (1) and phosphonate ester (2) ligands were studied by multinuclear (1H, 13C, 31P, and 17O) nuclear MR spectroscopy. RESULTS: The inner-sphere water lifetime in the Ln(2) complexes was much longer (tauM298 = 0.8-1.3 ms) than in the corresponding Ln(1) complexes. This allowed direct detection of the bound-water molecule in europium(2) in water at 40 degrees C by 1H nuclear MR. The water relaxivity of gadolinium(2) was independent of pH between 8.5 and 6.0, whereas the relaxivity of gadolinium(1) increased more than twofold in this pH range. CONCLUSIONS: T1-weighted images of phantoms containing gadolinium(1) at different pH values demonstrate the efficacy of this complex as a pH-sensitive MR contrast agent.
Assuntos
Meios de Contraste/química , Gadolínio/química , Imageamento por Ressonância Magnética , Humanos , Metais Terras Raras/química , ÁguaRESUMO
The distribution of magnesium in plasma, bound to proteins (pMg), complexed to low molecular weight anions (cMg) and ionized (iMg), was compared by size exclusion chromatography and an approach using a combination of atomic absorption spectroscopy, ion selective electrodes and 31P nuclear magnetic resonance spectroscopy. The distribution of pMg:cMg:iMg was 28:13:59 as determined by chromatography and 26:14:60 as determined by the latter methodology. These results are consistent with the hypothesis that plasma proteins and weak complexing anions correspond to high and low affinity magnesium binding ligands in plasma, respectively.
Assuntos
Cromatografia em Gel/métodos , Magnésio/sangue , Espectroscopia de Ressonância Magnética/métodos , Adulto , Cálcio/sangue , Feminino , Humanos , Pessoa de Meia-Idade , Valores de Referência , Reprodutibilidade dos TestesRESUMO
We present a simple (2)H NMR assay of the fractional contribution of gluconeogenesis to hepatic glucose output following ingestion of (2)H(2)O. The assay is based on the measurement of relative deuterium enrichment in hydrogens 2 and 3 of plasma glucose. Plasma glucose was enzymatically converted to gluconate, which displays fully resolved deuterium 2 and 3 resonances in its (2)H NMR spectrum at 14.1 T. The signal intensity of deuterium 3 relative to deuterium 2 in the gluconate derivative as quantitated by (2)H NMR was shown to provide a precise and accurate measurement of glucose enrichment in hydrogen 3 relative to hydrogen 2. This measurement was used to estimate the fractional contribution of gluconeogenesis to hepatic glucose output for two groups of rats; one group was fasted for 7 h and the other was fasted for 29 h. Rats were administered (2)H(2)O to enrich total body water to 5% over the last 4-5 h of each fasting period. For the 7-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.32 +/- 0.09 (n = 7). This indicates that gluconeogenesis contributed 32 +/- 9% of total hepatic glucose output with glycogenolysis contributing the remainder. For the 29-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.81 +/- 0.10 (n = 6), indicating that gluconeogenesis supplied the bulk of hepatic glucose output (81 +/- 10%).
Assuntos
Glicemia/metabolismo , Óxido de Deutério/farmacocinética , Gluconeogênese , Administração Oral , Animais , Glicemia/análise , Deutério , Óxido de Deutério/administração & dosagem , Espectroscopia de Ressonância Magnética/métodos , Masculino , Modelos Químicos , Técnica de Diluição de Radioisótopos , Ratos , Ratos Sprague-DawleyRESUMO
A kinetic model of the citric acid cycle for calculating oxygen consumption from (13)C nuclear magnetic resonance (NMR) multiplet data has been developed. Measured oxygen consumption (MVO(2)) was compared with MVO(2) predicted by the model with (13)C NMR data obtained from rat hearts perfused with glucose and either [2-(13)C]acetate or [3-(13)C]pyruvate. The accuracy of MVO(2) measured from three subsets of NMR data was compared: glutamate C-4 and C-3 resonance areas; the doublet C4D34 (expressed as a fraction of C-4 area); and C-4 and C-3 areas plus several multiplets of C-2, C-3, and C-4. MVO(2) determined by set 2 (C4D34 only) gave the same degree of accuracy as set 3 (complete data); both were superior to set 1 (C-4 and C-3 areas). Analysis of the latter suffers from the correlation between citric acid cycle flux and exchange between alpha-ketoglutarate and glutamate, resulting in greater error in estimating MVO(2). Analysis of C4D34 is less influenced by correlation between parameters, and this single measurement provides the best opportunity for a noninvasive measurement of oxygen consumption.
Assuntos
Ácido Glutâmico/análise , Espectroscopia de Ressonância Magnética/métodos , Miocárdio/metabolismo , Consumo de Oxigênio/fisiologia , Acetatos/farmacologia , Animais , Isótopos de Carbono , Ciclo do Ácido Cítrico/fisiologia , Glucose/farmacologia , Técnicas de Cultura de Órgãos , Consumo de Oxigênio/efeitos dos fármacos , Prótons , Ácido Pirúvico/farmacologia , Ratos , Análise de RegressãoRESUMO
The growth-inhibitory effect of cyclocreatine (CCr) and the kinetics of CCr and Na(+) cotransport were investigated in MCF7 human breast cancer cells and its adriamycin-resistant subline with use of (31)P- and (23)Na-NMR spectroscopy. The growth-inhibitory effect in the resistant line occurred at a lower CCr concentration and was more pronounced than in the wild-type line. This correlated with an approximately 10-fold higher affinity of CCr to the transporter in the resistant line. The passive diffusion coefficient of CCr was also higher in the resistant line by three- to fourfold. The transport of CCr was accompanied by a rapid increase in intracellular Na(+). This increase was found to depend on the rate of CCr transport and varied differently with CCr concentration in the two cell lines. It is proposed that the cotransport of CCr and Na(+) followed by increased Na(+) concentration, together with the accumulation of the highly charged phosphocyclocreatine, are responsible for cell swelling and death.
Assuntos
Neoplasias da Mama/metabolismo , Creatinina/análogos & derivados , Sódio/metabolismo , Transporte Biológico/fisiologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Divisão Celular , Creatina/farmacologia , Creatina Quinase/metabolismo , Creatinina/metabolismo , Creatinina/farmacologia , Feminino , Humanos , Membranas Intracelulares/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Fósforo , Células Tumorais CultivadasRESUMO
Most 13C nuclear magnetic resonance (NMR) isotopomer analyses relate a metabolic index of interest to populations of 13C isotopomers as reported by one-bond 13C-13C spin-spin couplings. Metabolic conditions that produce highly enriched citric acid cycle intermediates often lead to 13C NMR spectra of metabolites such as glutamate that show extra multiplets due to long-range couplings. It can be demonstrated from 13C NMR spectra of hearts perfused with mixtures of acetate plus propionate that multiplets in glutamate C2 arising from 3J25 coupling provide a direct readout of acetyl-CoA fractional enrichment (FC1 and FC3), while multiplets in glutamate C5 arising from 2J35 and 3J25 couplings quantitatively reflect enrichment of the anaplerotic substrate.