Loss of high-energy phosphate following hyperthermia demonstrated by in vivo 31P-nuclear magnetic resonance spectroscopy.

We have used in vivo 31P-nuclear magnetic resonance spectroscopy to study the changes in high-energy phosphates following hyperthermia. Immediately after heating, there is a fall in adenosine triphosphate and apparent intracellular pH and an increase in inorganic phosphate. Following sublethal heating (40 degrees for 15 min), these changes were partial, and they resolved over the subsequent 45 hr. With tumors given severe hyperthermia (47 degrees for 15 min), there was complete disappearance of adenosine triphosphate, with no recovery by 24 hr posttreatment. Qualitatively similar effects were seen after heating of normal leg muscle. The degree of fall of the adenosine triphosphate/inorganic phosphate concentration ratio was directly proportional to the heat dose and to thermal cell kill. 31P-Nuclear magnetic resonance spectroscopy may be useful in thermal dosimetry and treatment evaluation following hyperthermia.

Evidence for the presence of an ATP transport system in brush-border membrane vesicles isolated from the kidney cortex.

Results of 31P-NMR studies and transport experiments using the radioactive tracer technique are presented. They point to the conclusion that ATP is taken up into isolated renal brush-border membrane vesicles, possibly by a carrier-mediated mechanism.

Mössbauer, EPR and NMR studies of the acid-induced reduction and changes in spin state of ferric bleomycin.

Iron-57 Mössbauer, electron paramagnetic resonance (EPR) and H-1 nuclear magnetic resonance (NMR) studies of iron-bleomycin complexes in the pH range from 1.0 to 6.0 are reported. Sequential protonation of the ligands produces a variety of high-spin and low-spin complexes of the metal. Of particular interest is the reversible equilibrium between Fe(III)- and oxygen-stable Fe(II)-bleomycin. Below pH 3.5 Fe(II) complexes form, with maximal reduction occurring at approximately pH 2. At still lower pH, Fe(III) complexes unassociated with bleomycin become dominant. The observed reduction in the absence of exogenous reducing agents suggests the possible involvement of intramolecular autoreduction in bleomycin-mediated DNA degradation.

Proton nuclear magnetic resonance relaxation times in severe myocardial ischemia.

Contrast produced by differences in regional proton relaxation times (T1 and T2) provides the potential to assess the extent of myocardial infarction using nuclear magnetic resonance (NMR) imaging. Previous laboratory studies have shown that longitudinal (T1) and transverse (T2) relaxation times are prolonged in acute myocardial infarction, and these prolongations have been attributed entirely to increases in tissue water content. The present study seeks to evaluate the relation between both T1 and T2 and regional myocardial perfusion and water content throughout a wide range of blood flow reduction. The left anterior descending coronary artery and collateral vessels supplying a region of the anterior wall of the left ventricle were ligated in 10 dogs for 4 hours until they were killed. Both water proton and bulk proton relaxation times of myocardial samples from ischemic and control zones were measured at 200 and 20 MHz, respectively. Regions of severe ischemia (flow less than 5% of control) demonstrated no significant alteration in T1 compared with nonischemic myocardium. Greatest T1 and T2 elevations were observed in moderately ischemic myocardium (flow = 5 to 50% of control). The water relaxation behavior differed with the severity of the flow reduction and was not totally dependent on changes in water content. These data suggest that relaxation time alterations are more complex than previously reported in myocardial ischemic insult. In the future, using T1 weighted imaging methods, myocardial ischemic insults associated with severe reductions in blood flow would be anticipated to demonstrate a doughnut pattern with an area of abnormal intensity in the peripheral zone surrounding a central ischemic zone with normal intensity.

In vivo 31P nuclear magnetic resonance spectroscopy of human temporal lobe epilepsy.

We performed localized 31P nuclear magnetic resonance (NMR) 1H-image-guided in vivo spectroscopy to study regional high-energy phosphate levels in the brains of normal controls and in patients with intractable unilateral temporal lobe epilepsy. We did not observe differences in intracellular pH between controls and patients. The phosphocreatine/inorganic phosphate ratio was reduced by 50% in the epileptogenic temporal lobe compared with controls (p less than 0.005) and by 35% when compared with the unaffected contralateral temporal lobe (p less than 0.05). We did not observe differences in the ratio of phosphomonoesters to phosphodiesters between controls and patients. These findings suggest that in vivo 31P NMR spectroscopy yields a distinctive interictal metabolic profile in patients with intractable unilateral temporal lobe epilepsy and may permit noninvasive lateralizing evidence of the seizure focus.

Lateralization of human temporal lobe epilepsy by 31P NMR spectroscopic imaging at 4.1 T.

OBJECTIVE: To compare the phosphorous metabolite ratios in the mesial temporal lobe of healthy volunteers (n = 20) with the corresponding ratios in patients with temporal lobe epilepsy (n = 30) using 31P NMR spectroscopic imaging and to lateralize the seizure focus in temporal lobe epilepsy patients using various phosphorous metabolite ratios-phosphocreatine to inorganic phosphate (PCr/Pi), PCr to adenosine triphosphate (PCr/gamma-ATP), and (gamma-ATP/Pi)--and to compare with clinical lateralization results. METHODS: All 31P NMR spectroscopic imaging studies were performed on a high-field, 4.1 T, whole-body NMR spectroscopic imaging system using a 31P/1H double-tuned volume coil. RESULTS: We found an average reduction of 15% in the PCr/Pi and gamma-ATP/Pi ratios compared with the corresponding ratios in healthy volunteers in the entire mesial temporal lobe, and more than a 30% reduction in these two ratios in the anterior region of the epileptogenic mesial temporal lobe. These ratios were also reduced significantly in the ipsilateral lobe when compared with their corresponding values in the contralateral lobe. In patients we lateralized the seizure focus, based on these 31P NMR data, and compared the results with the clinical lateralization. The lateralization based on either the PCr/Pi or the gamma-ATP/Pi ratio yielded a correspondence of 70 to 73% with the final clinical lateralization. In the subgroup of patients (n = 9) that needed intracranial EEG for the presurgical lateralization because of inconclusive results from the noninvasive methods, a 78% correspondence was found with the 31P NMR-based lateralization, whereas MRI provided a correspondence of only 33%, and scalp EEG provided a correspondence of only 56%. CONCLUSIONS: These results suggest the utility of adding the 31P NMR method to the group of noninvasive modalities used for presurgical decision making in temporal lobe epilepsy patients.

Is the intracellular pH different from normal in the epileptic focus of patients with temporal lobe epilepsy? A 31P NMR study.

We performed in vivo 31P NMR spectroscopic studies of human brain on a 4.1 T whole-body NMR system. Based on a control group of 20 healthy volunteers, the normal pHi was 7.05 (SD, 0.06; SEM, 0.01) in the left temporal lobe and 7.04 (SD, 0.04; SEM, 0.01) in the right temporal lobe. We also studied a patient group consisting of 13 individuals with unilateral temporal lobe epilepsy. The mean pHi was 7.02 (SD, 0.04; SEM, 0.01) in the ipsilateral lobe and 7.02 (SD, 0.05; SEM, 0.01) in the contralateral lobe. These results clearly show that no statistically significant difference in pHi is observed between the two lobes, either in normal controls or in patients. Also, no significant pHi difference exists between the control group and the patient group. Lateralization in each of the 13 patients with unilateral epilepsy, based on their individual pHi difference between the ipsilateral lobe and contralateral lobe (delta pHi), showed that three patients were nondiagnostic cases because their delta pHis were not significantly different from zero (< or = 0.02), five patients showed small delta pHis consistent with their clinical lateralization, whereas the remaining five patients showed delta pHi-based lateralization opposite to the clinical findings. These results seem to indicate an essentially random distribution around delta pHi = 0 within a very small experimental error of +/-0.02 pH units. pHi obtained from eight different areas in each of the 13 unilateral patients also did not show any significantly nonzero delta pHi values. These results led to the conclusion that even at the excellent spectral resolution and reproducibility of the 4.1 T machine (typical SD of 0.05 pH units), no significant pHi effect, induced by temporal lobe epilepsy, could be detected. Therefore, in this study, delta pHi does not appear to be a clinically useful tool for the lateralization of epileptic foci in patients with temporal lobe epilepsy.

Synthesis and in vivo evaluation of new contrast agents for cardiac MRI.

Analogues 2-6 of N(3),N(6)-bis(2'-myristoyloxyethyl)-1, 8-dioxotriethylenetetraamine-N,N,N',N'-tetraacetic acid (BME-DTTA) (1), which like 1 are also based on the DTTA structure but contain shorter fatty acyl chains, were synthesized to improve the water solubility of the corresponding gadolinium complexes. The gadolinium complexes of 1 and 3-5 have very low solubility in water. Thus liposomal preparations are necessary for their in vivo MRI application. These liposomal preparations retain high in vitro relaxivities (27.1, 21.57, 20.32, 23.1 s(-1) mM(-1), respectively) and induce sustained MRI signal intensity enhancements (67.2, 38.4, 52.1, 41.7 in arbitrary units, respectively). The gadolinium complex of 2 is quite soluble in water. Its lifetime in the blood stream, however, is short. The gadolinium complex of analogue 6, N-(2-butyryloxyethyl)-N'-(2-ethyloxyethyl)-N,N'-bis[N' ',N' '-bis(carboxymethyl)acetamido]-1,2-ethanediamine (ABE-DTTA), has demonstrated its potential as a water-soluble, cardiac-specific, MRI contrast agent. It is completely soluble in water at a 25 mM concentration, allowing the preparation of an injectable dose. The in vitro relaxivity of the complex is 16.24 s(-1) mM(-1). The agent shows a specific accumulation in the heart tissue reaching its maximum within 15 min after administration, inducing a sustained MRI signal intensity enhancement of 43.6%. This enhancement lasts for at least 3 h, thus indicating a reasonably long lifetime of this contrast agent in the myocardium without deleterious effects on heart function parameters.

Shift-reagent-aided 23Na NMR spectroscopy.

Nondestructive observation of intracellular sodium (Na+i) levels is of utmost clinical and biochemical importance, 23Na is a relatively sensitive nuclear magnetic resonance (NMR)-observable nuclide, and NMR is intrinsically noninvasive. Since, however, the frequency position of Nai+ and Nao+ is identical, paramagnetic dysprosium complexes have to be used as shift reagents. The latter differentiate between signals from intracellular and extracellular spaces, providing a nondestructive, continuous method to monitor intracellular cation concentration, in real time. In this article, the methodology of shift-reagent-aided 23Na NMR spectroscopy is discussed. Application is demonstrated by results from two studies in isolated perfused rat hearts. In hearts subjected to hypoxia (or ischemia) followed by reoxygenation (or reflow), the authors show that recovery following an ischemic or hypoxic insult can be predicted by monitoring Na+i levels by NMR. In a study in paced hearts, the authors show an accumulation of Na+i with increased heart rate, and also a positive coupling between elevated sodium levels and increased systolic pressure during the pacing-up period, as predicted by the Na-pumplag theory.

Studies on bleomycin-DNA and bleomycin-iron interactions.

The bleomycins, a group of antitumor antibiotics (Figure 1), cause the degradation of DNA by a process requiring iron(II) and dioxygen (1,2). DNA degradation appears to involve two steps: association of the drug with the nucleic acid and degradation of the DNA. As part of studies directed toward achieving an understanding of how the bleomycins degrade DNA, we have examined various properties of the drug using a variety of chemical and physico-chemical techniques, including NMR and Mössbauer spectroscopy. We have studied both the interaction of the antibiotic with its target (DNA) as well as its association with its metal ion cofactor. This work has been performed on the intact drug and its derivatives as well as on synthetic models of the parent drug. This paper reviews and updates the recent work from this laboratory on the bleomycins.

In vivo R1-enhancement mapping of canine myocardium using ceMRI with Gd(ABE-DTTA) in an acute ischemia-reperfusion model.

PURPOSE: To demonstrate the usefulness of normalized DeltaR1 (DeltaR1(n)) mapping in myocardial tissue following the administration of the contrast agent (CA) Gd(ABE-DTTA). MATERIALS AND METHODS: Ischemia-reperfusion experiments were carried out in 11 dogs. The method exploited the relatively long tissue lifetime of Gd(ABE-DTTA), and thus no fast R1 measurement technique was needed. Myocardial perfusion was determined with colored microspheres (MP). RESULTS: With varying extent of ischemia, impaired wall motion (WM) and lower DeltaR1(n) values were detected in the ischemic sectors, as opposed to the nonischemic sectors where normal WM and higher DeltaR1(n) were observed. Based on the DeltaR1(n), data from the myocardial perfusion assay and the DeltaR1(n) maps were compared in the ischemic sectors. A correlation analysis of these two parameters demonstrated a significant correlation (R = 0.694, P < 0.005), validating the DeltaR1(n)-mapping method for the quantitation of ischemia. Similarly, pairwise correlations were found for the MP, DeltaR1(n), and wall thickening (WT) values in the same areas. Based on the correlation between DeltaR1(n) and MP, DeltaR1(n) maps calculated with a pixel-by-pixel resolution can be converted to similarly high-resolution myocardial perfusion maps. CONCLUSION: These results suggest that the extent of the severity of ischemia can be quantitatively represented by DeltaR1(n) maps obtained in the presence of our CA.

Gadolinium and dysprosium chelates of DTPA-amide-dextran: synthesis, 1H NMR relaxivity, and induced 23Na NMR shift.

In this study the conjugated macromolecular ligand, diethylenetriaminepentaacetic acid (DTPA)-amide-dextran, was synthesized by attaching DTPA to the dextran macromolecule (M(r) approximately 6000) by a covalent amide bond. Subsequently, DTPA-amide-dextran was complexed with either of the two lanthanide metal ions dysprosium (Dy) or gadolinium (Gd). The paramagnetic 23Na NMR shift induced by Dy(DTPA-amide-dextran) and the relaxivity (rho 1) induced by Gd(DTPA-amide-dextran) were characterized. Dy(DTPA-amide-dextran) induced a 25% larger 23Na NMR shift than that induced by Dy(DTPA). Neither the shift induced by Dy(DTPA-amide-dextran) nor the shift induced by Dy(DTPA) was affected by increasing levels of calcium ions in the solution. offDTPA-amide-dextran) exhibited an in vitro rho 1 of 8.4 (mM s)-1 at a 0.23 T magnetic field and 9.3 (mM s)-1 at a 0.47 T magnetic field, thus indicating a positive magnetic field dependence.

Evidence from 23Na NMR studies for the existence of sodium-channels in the brush border membrane of the renal proximal tubule.

A fast Na+-exchange is shown to be present in isolated renal brush border membranes. The lower limit of the rate constant for this process, calculated from the 23Na-NMR spectrum is 580 sec-1. The actual exchange rate may be higher. A fast 7Li exchange is also shown to be present in the isolated membrane vesicles. The characteristic overshoot of the Na+ dependent D-glucose cotransport and Na+/H+ antiport can be demonstrated. The fact that neither treatment with papain, nor lowering of the temperature to 5 degrees C affected the 23Na-NMR spectra obtained in the renal brush border membrane vesicles is consistent with the possibility that the fast Na+-exchange occurs through a channel mechanism.

In vitro ethanol effects on the transport properties of isolated renal brush-border membrane vesicles.

The in vitro effect of ethanol on membrane structure and transport properties was studied in isolated renal brush border membrane vesicles. 31P-NMR studies showed a dose-dependent increase in the quantity of an isotropic, possibly inverted-micellar component of the renal brush-border membrane as a result of treatment with ethanol. Such structures have been shown to be instrumental in the translocation of material across membrane bilayers. A 23Na-NMR study of Na+ exchange in artificial phosphatidylcholine liposomes indicated that ethanol (0.1%) was capable of rendering the otherwise inert vesicles permeable to sodium, supporting the idea that ethanol may exert its action via a direct effect on the structure of the phospholipid bilayer. In the isolated renal brush-border membrane vesicles, like in the artificial liposomes, amiloride-insensitive pathways of Na+ transport were shown to be markedly activated by ethanol. These results were consistent with the inhibitory effect ethanol had on Na+ gradient-dependent transport systems such as the Na+ gradient-dependent D-glucose transport and Na+/H+ exchange. In conclusion, our results indicate that ethanol exerts its effect on the renal brush-border membrane by causing a structural change in the phospholipid bilayer which activates sodium intake. The inhibitory effect of ethanol on glucose uptake and Na+/H+ exchange is secondary, as a result of the dissipation of the energy-producing Na+ gradient.

Catalysis of oxygen-18 exchange between inorganic phosphate and water by the gastric H,K-ATPase.

The gastric H,K-ATPase is shown to catalyze 18O exchange between Pi and HOH. Mg2+ is the only ion required for the reaction. K+ increases the rate of isotope exchange, which is directly proportional to specific ATPase activity. Ouabain, which potently inhibits the Na,K-ATPase, has no effect on the exchange reaction. Conversely, omeprazole, which is specific for the H,K-ATPase, completely inhibits 18O exchange. Vanadate inhibition of exchange can be explained by competitive binding with Pi. The rate of 18O exchange is faster than the hydrolytic rate and about equal to the dephosphorylation rate. Thus, the ionic requirements for exchange, inhibition of exchange, and the rate of exchange are all compatible with catalysis occurring via the same phosphoenzyme intermediate formed during hydrolysis of ATP. The distribution of 18O-labeled Pi species formed with time indicates that Pi loss is only about twice as fast as covalent bond formation. This kinetic pattern is unaffected by K+, temperature, or the specific activity of the enzyme preparation. Invariance of the kinetic pattern could mean isotope exchange is always catalyzed by the same form of the enzyme, and K+ and higher temperature accelerate the reaction by increasing the relative amount of the active conformer. Independence of the kinetic pattern from specific activity implies that the catalytic mechanism of active enzyme molecules is unaffected by inactive proteins in gastric microsomal membranes.

Fatty-acyl iminopolycarboxylates: lipophilic bifunctional contrast agents for NMR imaging.

New fatty-acyl contrast agents, N3-2'-myristoyloxyethyl-N6-2'-hydroxyethyl-1, 8-dioxotriethylenetetraamine-N,N,N',N'-tetraacetic acid (MHE-DTTA) and N3,N6-bis(2'-myristoyloxyethyl)-1,8-dioxo- triethylenetetraamine-N,N,N',N'-tetraacetic acid (BME-DTTA) were prepared by sequential alkylation, acylation, and catalytic hydrogenation from bis(hydroxyethyl)-ethylenediamine with satisfactory yields (overall 36-46%). The 1:1 gadolinium complexes of the ligands MHE-DTTA and BME-DTTA were incorporated into liposomes and their relaxivities in vitro were determined. The relaxivities of both agents were similar and were greater than those of Gd3+ aquoion, Gd(EDTA), and Gd(DTPA) at both 0.23 T and 0.47 T. The relaxivities of these two agents increased from the lower to the higher magnetic field, indicating a positive field dependence. This is advantageous because of the widespread use of high-field (B0 greater than 0.5 T) NMR imaging instruments. Stability constants (log K) of Gd(MHE-DTTA) and Gd(BME-DTTA) were found to be 15.27 +/- 2.21 and 16.78 +/- 0.36, respectively. LD50 of both compounds was greater than 0.2 mmol/kg. These stabilities and lower limits of LD50 indicate the possible in vivo application of these agents.

In vivo characterization of Gd(BME-DTTA), a myocardial MRI contrast agent: tissue distribution of its MRI intensity enhancement, and its effect on heart function.

We have determined an LD50 of 0.56 +/- 0.05 mmol/kg for liposomal Gd(BME-DTTA) in mice and also shown that liposomal Gd(BME-DTTA) has no deleterious effects on heart rate, blood pressure, left ventricular force and AV conductance in ferret hearts in vivo at the magnetic resonance imaging (MRI)-effective dose of 0.05 mmol/kg body weight. In MRI images, a 1H signal intensity enhancement is observed in the following organs in decreasing order of the effect: heart approximately spleen > kidney > liver. This enhancement is stable for over 3 h in all organs. The results of 1H MRI and electron micrographs indicate that the lipophilic fatty acyl groups in the ligand BME structure and the particle sizes of liposomal Gd(BME-DTTA) are two important factors for tissue specificity of liposomal Gd(BME-DTTA) in the intensity enhancement. In vitro relaxivity of a liposomal Gd(BME-DTTA) sample, stored at 4 degrees C, remained stable for over 4 months of observation, but a significant decrease in relaxivity was observed in a sample stored at room temperature, most likely reflecting some deterioration in liposome chemistry.