1H Spin-echo NMR Spectroscopy Research Articles

19F NMR spectroscopy study of the metabolites of flucloxacillin in rat urine.

19F NMR spectroscopy was used to monitor the metabolites of flucloxacillin in the urine of a rat dosed with its sodium salt. 19F NMR signals were detected and quantified from flucloxacillin and three metabolites. The 19F NMR method involves minimal sample preparation and is free from interference by endogenous urine components. High-field spin-echo 1H NMR spectroscopy and HPLC were used to confirm the results.

Phase equilibria and physical-chemical properties of sugar-based surfactants in aqueous solutions

A study of solution properties of 3 nonionic surfactants derived from glucose is reported. These surfactants, from a series of 6- O-acyl- α, β- d-glucopyranoses, differ from one other by the number of carbon atoms in their alkyl chain: 6- O-decanoyl-, 6- O-stearoyl- and 6- O-oleyl-. The surface properties [critical micellar concentration (cmc) and minimal area/molecule at air-water interface (a min)] were investigated in water at 42 °C by means of surface tension measurements. Partial phase diagrams and liquid-crystalline mesophases were studied in heavy water using polarising microscopy, 2H-NMR spectra and DSC measurements. All binary systems form a lamellar lyomesophase in a wide range of composition. A Gel → Lamellar phase transition was observed as the temperature was increased only on the 6- O-decanoyl- d-glucopyranose/D 2O system. The Gel-Lamellar transition temperatures were checked by DSC thermograms. Molecular self-diffusion of surfactant and water in the micellar solution phase of 6- O-decanoyl- d-glucopyranose/D 2O system were determined at 35 °C by Fourier transform pulsed gradient spin-echo 1H-NMR spectroscopy. From this study it has been possible to draw conclusions about the changes on the solution microstructures as function of surfactant concentration.

Diffusional Behavior ofn-Alkanes in Polypeptide Gel System with Highly Oriented Chains As Studied by Pulsed Field Gradient Spin Echo1H NMR Method

The diffusion coefficients (D) of n-alkanes, n-pentane and n-decane, and 1,4-dioxane as solvent in poly(γ-benzyl l-glutamate) (PBLG) gel with highly oriented α-helical chains were measured by pulsed field gradient spin echo 1H NMR spectroscopy, of which the structure was determined by X-ray diffraction. From the experimental results, it was found that the diffusion coefficients of n-pentane and n-decane in the PBLG gel for the direction parallel and perpendicular to the magnetic field are D∥ = 8.4 × 10-6 cm2/s and D⊥ = 6.4 × 10-6 cm2/s, respectively, and D∥ = 6.1 × 10-6 cm2/s and D⊥ = 5.0 × 10-6 cm2/s, respectively, which correspond to the directions parallel and perpendicular to the α-helical main chain.

Hepatic [2-13C]acetate metabolism by jirds infected with Echinococcus multilocularis

Carbon-13 decoupled 1H spin echo NMR spectroscopy, with and without population inversion, was used to study carbon flow between the host, Meriones unguiculatus, and the parasite, Echinococcus multilocularis. This was accomplished by monitoring [2-13C]acetate metabolism in the liver of jirds infected with metacestodes of this parasite. Thirty minutes after injection of labelled acetate solution into the portal vein, 13C enrichment was observed in hepatic acetate, β–hydroxybutyrate, succinate, alanine, lactate and glucose. For E. multilocularis cysts, at this time,13C enrichment was observed in the same metabolites as in livers and, in addition, citrate. At 120 min there was a significant decrease in the amount of label present in all hepatic metabolites whereas more label was found in the majority of the parasite metabolites. The results confirm that exogenous acetate, through randomization of the 13C in biochemical pathways of host liver, ends up in hepatic glucose. As this biosynthetic route is not available to the parasite, the presence of 13C enriched glucose in the cysts clearly indicates that the parasite is siphoning off glucose that is newly synthesized by the host. At 120 min some of this labelled glucose was stored in parasite glycogen whereas some of it had been catabolized to succinate, alanine, lactate and acetate, end products which are excreted back into the host.

Hepatic [2-13C]acetate metabolism by jirds infected with Echinococcus multilocularis

Carbon-13 decoupled 1H spin echo NMR spectroscopy, with and without population inversion, was used to study carbon flow between the host, Meriones unguiculatus, and the parasite, Echinococcus multilocularis. This was accomplished by monitoring [2-13C]acetate metabolism in the liver of jirds infected with metacestodes of this parasite. Thirty minutes after injection of labelled acetate solution into the portal vein, 13C enrichment was observed in hepatic acetate, β–hydroxybutyrate, succinate, alanine, lactate and glucose. For E. multilocularis cysts, at this time,13C enrichment was observed in the same metabolites as in livers and, in addition, citrate. At 120 min there was a significant decrease in the amount of label present in all hepatic metabolites whereas more label was found in the majority of the parasite metabolites. The results confirm that exogenous acetate, through randomization of the 13C in biochemical pathways of host liver, ends up in hepatic glucose. As this biosynthetic route is not available to the parasite, the presence of 13C enriched glucose in the cysts clearly indicates that the parasite is siphoning off glucose that is newly synthesized by the host. At 120 min some of this labelled glucose was stored in parasite glycogen whereas some of it had been catabolized to succinate, alanine, lactate and acetate, end products which are excreted back into the host.

Raised transaminase activity of blood plasma from rats with experimentally-induced kidney damage detected by spin-echo 1H-NMR spectroscopy

We report the application of spin-echo 1H-NMR spectroscopy to the detection of raised plasma transaminase activity in rats treated with the nephrotoxic cephalosporin antibiotic cephaloridine (CPH). Spin-echo 1H-NMR analysis of lyophilized plasma, reconstituted in H 20 reveals a doublet at δ 1.48 for alanine. However when samples were reconstituted with 2H 20 we noted that in samples from CPH-treated rats (but not in control samples) there was a variable degree of appearance of a singlet at δ 1.47 together with a reduction in the doublet at δ 1.48. We suggest that this is due to the release of transaminases from damaged tissue which, via a reversible conversion of alanine to pyruvate, causes selective deuteration of alanine at the α-hydrogen (α-CH) position. This observation suggests that these 1H-NMR spectral patterns are dependent on the level of plasma transaminases and this may provide a novel indicator of tissue damage.

Studies of oxidative stress in cellular systems The interaction of monocytes and erythrocytes

1H spin echo NMR spectroscopy is used to follow the interaction of intact and viable erythrocytes and monocytes obtained from different sources in mixed cultures. After a lag time (270 min) erythrocyte glutathione is observed to become more oxidised. This result is believed to occur as a consequence of monocyte activation generating hydrogen peroxide or hypochlorous acid, which is targeted at the erythrocyte. The red cell in turn employs its sulphydryl system as an anti-oxidant defence.

Clinical analysis in intact erythrocytes using 1H spin echo NMR

A new method of clinical analysis based on 1H spin echo NMR spectroscopy is presented. It is capable of providing information on six metabolites within viable erythrocytes, directly and without any preparative procedures prior to analysis except for cell separation and washing. Erythrocytes from patients with rheumatoid arthritis and Graves' disease are compared with cells obtained from healthy volunteers. The NMR detectable species in the cytosol of the cells are glutathione, ergothioneine, choline, creatine, glycine, lactate and to a lesser extent alanine and valine. Significant differences are observed between the ergothioneine pools in the rheumatoid group ( P < 0.01) compared to the control group. The glutathione: di-glutathione ratio can be assessed from the ratio, g 2 to g 4, taken from different signals in the glutathione molecule. The total concentration of glutathione present is easily assessed qualitatively but is more difficult to quantitate.

A quantitative study of the complexation of cadmium in hemolyzed human erythrocytes by1H NMR spectroscopy

The stability of complexes formed by Cd2+ in hemolyzed human erythrocytes was studied by spin-echo 1H NMR spectroscopy. Changes in resonances for the carbon-bonded protons of glutathione (GSH) upon addition of the ethylenediaminetetraacetic acid complex of Cd2+ (Cd(EDTA)2-) and the appearance of resonances for Mg(EDTA)2- indicate that the Cd(EDTA)2- complex dissociates in hemolyzed erythrocytes with the formation of Cd(GSH)x and Mg(EDTA)2- complexes. A semiquantitative estimate of the overall stability constant for the complexation of Cd2+ in hemolyzed erythrocytes was obtained from spin-echo 1H NMR data. The stability constant is consistent with the majority of the Cd2+ in erythrocytes present as Cd(SG)2(2-). A conditional equilibrium constant was also determined for the complexation of Mg2+ by ligands in hemolyzed human erythrocytes.

Demonstration of oxidative stress across cell membranes in chronic heart failure using 1H spin echo NMR spectroscopy of intact erythrocytes

Demonstration of oxidative stress across cell membranes in chronic heart failure using 1H spin echo NMR spectroscopy of intact erythrocytes

An NMR compatible model for solid tumour

An NMR model for the study of drug interactions with solid tumour is described. It is based on the use of packed cells in a simple medium. The system is capable of dealing with cellular biochemistry at a molecular level through experiments conducted in a homogeneous field by 1H spin-echo NMR spectroscopy. However, through a simple imaging technique, it is also possible to introduce a spatial element into the chemistry of the packed cells. The use of these complementary NMR methods are illustrated by the action of the cytotoxic agent doxorubicin on cellular glycolysis. A pattern of signals observed in a homogeneous field experiment following oxidative stress are duplicated in the imaging experiment to illustrate the toxins' ability to penetrate the model tumour.

The oxidation of glutathione by nitroprusside: Changes in glutathione in intact erythrocytes during incubation with sodium nitroprusside as detected by 1H spin echo NMR spectroscopy

Spin echo 1H NMR spectroscopy showed that when the hypotensive agent sodium nitroprusside, Na 2[Fe(CN) 5NO]·2H 2O, was incubated with intact erythrocytes in 2H 2O saline, glutathione in the erythrocytes was oxidised to diglutathione. This was confirmed by 1H FT NMR for the in vitro reaction. 13C FT NMR showed that the stoichiometry of the glutathione nitroprusside reaction was 1:1; the inorganic products were nitric oxide and hexacyanoferrate(II), [Fe(CN) 6] − . At no stage was free cyanide liberated. The reaction of nitroprusside with glutathione, which occurs after the nitroprusside has crossed the erythrocyte membrane, is compared with the reaction of nitroprusside with haemoglobin. In neither of these reactions with major erythrocyte components was any free cyanide liberated by sodium nitroprusside.

A 1H spin echo NMR study of the HeLa tumour cell

Phosphorylcholine, phosphorylcreatine, lactate, glutathione, glycine and leucine were identified in living HeLa cells in the first study of this cell type by 1H spin echo NMR spectroscopy. The technique has the advantage that it is non-invasive, providing detailed structural information on individual species present in the cell matrix. It has been used in this case to study the rate of energy consumption following the activation of the glycolytic pathway with glucose.

Changes in glutathione in intact erythrocytes during incubation with penicillamine as detected by 1H spin-echo NMR spectroscopy

1H spin-echo NMR spectroscopy was used to study changes in glutathione status in intact erythrocytes. The concentration of glutathione in suspensions of erythrocytes in 2H 2O saline is significantly different in cells from patients with rheumatoid arthritis than in controls from normal healthy volunteers. It is observed that the methods of separation and suspension affect lactate metabolism in red cells. Incubation of erythrocytes with solutions of the therapeutic agent d-penicillamine in 2H 2O saline produced a change in glutathione resonances which is indicative of an increase in diglutathione concentration. Signals from the methyl groups of penicillamine decreased at a commensurate rate. Incubation of normal cells with plasma from patients with rheumatoid arthritis who had the same blood group as the normal volunteer indicated a much larger fall in glutathione signal with plasma from a patient treated with penicillamine than from a patient on non-steroidal anti-inflammatories.

Application of progress curve analysis to in situ enzyme kinetics using 1H NMR spectroscopy

The steady-state kinetics of enzymes in tissues, cells, and concentrated lysates can be characterized using high-resolution nuclear magnetic resonance spectroscopy; this is possible because almost invariably there are differences in the spectra of substrates and products of a reaction and these spectra are obtainable even from optically opaque samples. We used 1H spin-echo NMR spectroscopy to study the hydrolysis of α- l-glutamyl- l-alanine by cytosolic peptidases of lysed human erythrocytes. Nonlinear regression of the integrated Michaelis-Menten expression onto the progress-curve data yielded, directly, estimates of V max and K m for the hydrolase; a procedure for analyzing progress curves in this manner was adapted and compared with a commonly used procedure which employs the Newton-Raphson algorithm. We also performed a sensitivity analysis of the integrated Michaelis-Menten expression; this yielded equations that indicate under what conditions estimates of K m and V max are most sensitive to variations in experimental observables. Specifically, we showed that the most accurate estimates of the steady-state parameters from analysis of progress curves are obtained when the initial substrate concentration is much greater than K m . Furthermore, estimates of these parameters obtained by such an analysis are most sensitive to data obtained when the reaction is 60–80% complete, having started with the highest practicable initial substrate concentration.

Enkephalin degradation by human erythrocytes and hemolysates studied using 1H NMR spectroscopy

High resolution (400 MHz) 1H spin-echo NMR spectroscopy was used to monitor the degradation of leucine-enkephalin, and peptide fragments of it, by human erythrocytes and hemolysates. We showed that leucine-enkephalin is rapidly degraded by the cytosolic peptidases of the human erythrocyte, and we have elucidated the most probable pathway of degradation. Computer simulations of the proposed pathway, using a model incorporating the experimentally derived steady-state kinetic parameters obtained for the individual enzyme steps, showed close agreement with the experimental results. From a methodological perspective, the work demonstrates the value of 1H spin-echo NMR spectroscopy for rapidly elucidating, both qualitatively and quantitatively, an entire peptide-degradation pathway as it operates in situ.

The assimilation of tri- and tetrapeptides by human erythrocytes

Evidence is presented that tripeptides enter human erythrocytes via saturable transport system(s) at rates similar to those previously described for dipeptides (King, G.F. and Kuchel, P.W. (1985) Biochem. J. 227, 833–842) but that the transmembrane flux rates for tetrapeptides are considerably less. 1H spin-echo NMR spectroscopy was used to monitor the coupled uptake and hydrolysis of peptides by red cells, since it enabled the simultaneous measurement of the levels of substrates and products of peptidase-catalysed reactions in suspensions with haematocrits similar to those found in vivo. Weighted non-linear least-squares regression of the integrated Michaelis-Menten equation onto progress curves obtained from the hydrolysis of Tyr-Gly-Gly and Gly-Gly-Gly in RBC lysates gave K m = 2.11 ± 0.08 and 23.4 ± 0.9 mmol/l and V max = 307 ± 3 and 905 ± 22 mmol/h per l packed cells, respectively. In whole cell suspensions, the rate of hydrolysis was considerably less and was dominated by the transmembrane flux of tripeptide. Progress curve analysis thus yielded the steady-state kinetic parameters for peptide transport; the values were K m = 11.6 ± 1.1 and 56 ± 18 mmol/l and V max = 12.9 ± 3.0 and 36.4 ± 3.2 mmol/h per l packed cells, respectively, for the previously mentioned peptides. The rate of transport of the tetrapeptide Gly-Gly-Gly-Gly was considerably less than either of the tripeptides. The abovementioned steady-state kinetic parameters were used in computer simulations of the coupled uptake and hydrolysis of tripeptides by human erythrocytes under physiological conditions; these simulations revealed certain similarities between the rates of peptide uptake by erythrocytes and the intestine in vivo.

Proton NMR spectroscopic studies of dipeptidase in human erythrocytes

In studies on human erythrocyte metabolism in situ, high resolution (400 MHz) 1H spin-echo NMR spectroscopy was used to follow the time dependence of hydrolysis of glycylglycine and L-cysteinylglycine in intact cells and their lysates. The concentration dependence of the hydrolysis of L-cysteinylglycine was described by a rectangular hyperbola with K m, 3.5 ± 0.6 mmol/llysate and V max, 64.2 ± 3.2 mmol/llysate/h. We demonstrated that glycylglycine readily enters the erythrocyte and we introduce a means of analysing the data from the coupled reaction sequence; the sequence consists of transport followed by enzyme catalysed hydrolysis.

Endogenous phospholipase and choline release in human erythrocytes: A study using 1H NMR spectroscopy

1H spin-echo NMR spectroscopy has been used to show for the first time, to our knowledge, the presence of a phospholipase D activity in human erythrocytes. The enzyme is presumably intra-cellular and we have demonstrated that it is activated by a component of Krebs bicarbonate medium; most probably calcium ions. The existence of a phospholipase in human erythrocytes allows a simple explanation for the choline accumulation that occurs in these cells of manic-depressive and other patients receiving lithium carbonate therapy.