Alice M. Wyrwicz

In vivo 19F-NMR study of isoflurane elimination from brain

The time course of isoflurane elimination from rabbit brain was studied in vivo with 19F-NMR spectroscopy. Two exponential decay functions with different time constants were observed and assigned to two distinct brain compartments. Isoflurane has a 26 min time constant for one compartment (similar to a value of 25 min with halothane) but 174 min in the second one, compared with 320 min for halothane. The shorter half-life for isoflurane may be due to lower solubility of this agent in brain tissue. Comparison of isoflurane 19F chemical shifts in solvents in isolated brain lipids and in whole brain tissue indicates that the anesthetic present in the brain exists in a single environment (on the NMR time scale), which is a weighted average of both hydrophilic and hydrophobic environments.

In vivo 19F-NMR study of halothane distribution in brain

Halothane distribution and elimination from rabbit brain was studied in vivo using 19F-NMR spectroscopy. Two exponential decay functions for the anesthetic were observed in the clearance curve. They are assigned to halothane in brain held in two distinct chemical environments characterized by different chemical shifts, and half-lives (25 and 320 min). A nonvolatile halothane metabolite with a half-life of several days was found to be present in rabbit brains. The in vivo results were corroborated by ex vivo experiments on excised brain tissue. Halothane was distributed in all of the major cell subfractions, whereas the metabolite was present predominantly in the cytoplasm.

31P nmr study on the binding of 3′-cytidine monophosphate to ribonuclease A. Part I

Abstract An nmr chemical exchange method has been used to determine the pH dependence of the 31 P chemical shifts of the 3′-cytidine monophosphate·RNase A complex. The results are in conflict with a related 31 P nmr study by Lee and Chan and we suggest that these differences may be attributed to extraneous heavy metal ion impurities in their samples. The 31 P nmr titration data confirm that the dianionic phosphate inhibitor binds to a diprotonated active site and two ionizations have been observed with pK 1 = 4.4 and pK 2 = 6.2. A detailed description of the ionic states involved in the binding process is presented.

Multiple environments of fluorinated anesthetics in intact tissues observed with 19F NMR spectroscopy

The incorporation of two fluorine‐containing general anesthetic agents, halothane and methoxyflurane, into erythrocytes (from three different species), rabbit muscle and rabbit nerve, was followed with 19F NMR spectroscopy. Two major findings emerged from these studies: (1) multiple environments indicative of domain structure in the membrane can be observed depending on the anesthetic and the tissue type; and (2) the 19F chemical shifts of a given anesthetic were characteristic for the tissue examined. Halothane showed a single resonance in erythrocytes and multiple resonances in muscle and nerve, while methoxyflurane showed multiple resonances in both muscle and erythrocytes. The range of the 19F chemical shifts for the multiple peaks was as great as 6 ppm.

A 31P NMR study of phosphoenolpyruvate transport across the human erythrocyte membrane

Summary Intracellular PEP signals were noted in 31P NMR spectra obtained directly from intact erythrocytes when (i) red cells were suspended in 0.1 M citrate buffer (pH 6.0), (ii) glycolysis was inhibited with 10 mM NaF, (iii) extracellular PEP signals were broadened by Mn ion (1 mM), and (iv) a high medium PEP concentration (65 mM) was used to assure significant PEP uptake over short periods of signal accumulation. In the absence of NaF, PEP signals were not observed but 2,3-bisphosphoglycerate concentration increased at three times the PEP entry rate in NaF-poisoned cells. This study demonstrates that PEP transport into intact erythrocytes and associated metabolic sequelae can be monitored noninvasively with the aid of 31P NMR.

31P nuclear magnetic resonance observations in biological systems I. Intact tissue

Recent technological advances in nmr have pushed the practically observable concen- tration for phosphorus nuclei into the mil- limolar realm. As an example, a 100-90 MHz machine (refers to the frequency at which protons (‘H) resonate) using Fourier transform technology, can measure con- centrations of 0.3 mM overnight. With the addition of wide bore superconducting sys- tems, which can use tubes up to 20 mm in diameter, 1.0 mM concentrations can be detected in a few minutes. Because these are concentrations of interest in biological sciences, there has been a burst of studies of phosphorus nmr in biochemical systems. Greatest interest has arisen not just because of technological advances but also because recent observations have proven that whole tissue can give excellent spectra. Since sample inhomogeneity can lead to broadening of signals and make interpreta- tion of spectra difficult, one would be tempted to predict that whole tissue would give very broad signals. However, Moon and Richards [ 11 reported well-resolved *

1H Nmr study on the binding of CMP inhibitors to RNase A. III. Chemical exchange and relaxation effects

Summary A new dynamic 1 H nmr method has been developed and applied to the binding of 3′- and 5′-cytidine monophosphate (CMP) to Ribonuclease A. The study has provided information on the rates of chemical exchange and the degree of inhibitor immobilization at the active site. Evidence is presented which indicates that exchange-broadening effects on the line widths result from a slow pH-dependent conformational change of the enzyme inhibitor complex. At pH 4.5–5.5 and 24° 1/τ∼150–250 sec −1 for the CMP inhibitors with the exchange rate increasing with increasing pH. The rotational correlation time for the pyrimidine ring of the complex is 4.4 × 10 −9 see and for the ribose ring of the complex is 1.2 × 10 −8 sec. These results indicate that the ribose ring is rigidly bound to the enzyme while there remains some residual degree of rotational freedom about the glycosidic bond.

19F NMR imaging of a fluorinated anesthetic in a model system

Previous fluorine-19 NMR imaging studies have focused primarily on potential applications of perhuorocarbon liquids (Z-3) and emulsions (4-6) as contrast agents for a variety of organs. One of the disadvantages in using perfluorinated agents for fluorine imaging is the multiplicity of resonances arising from chemically nonequivalent fluorine atoms. These widely spaced resonances (50150 ppm) introduce chemicalshift artifacts in the frequency-encoded spatial dimension and require a spectrum simplification (7) prior to image collection. The published in vivo i9F NMR studies of fluorinated anesthetics (8, 9), fluorinated glucose analogs (l&13), and fluorouracil(14, 15) have employed primarily spectroscopic analysis, rather than imaging techniques. The one-dimensional 19F mapping of the fluorinated anesthetic distribution in rat brain showed it to be nonuniform and time-dependent, but did not provide sufficient anatomical detail to assign the signals to known structures (16). Assessment of drug delivery and drug biodistribution provides a different, as yet unexplored, area for potential i9F imaging application. In this communication we demonstrate the parallel use of 19F with ‘H NMR imaging to determine the distribution of fluorinated drugs in biological tissues. As an example we chose the general anesthetic halothane (CF

Proton nuclear magnetic resonance spectroscopy of plasma lipoproteins in head and neck cancer patients

HBrCl), which has a high lipid solubility, in the model host system provided by a green pepper. The distribution of halothane in a green pepper equilibrated with a saturated (15 n&f) aqueous solution of the anesthetic is illustrated in Fig. 1, which shows fluorine-19 (upper) and proton (lower) images of the same cross section. Halothane partitions preferentially into hydrophobic environments and thus is found in the oilcontaining seeds of the pepper. The proton image helps to define the structures within the pepper which contain this fluorinated compound. Both images are of the 5 mm thick slice obtained with a 6 in. imaging coil tuned to the fluorine-19 frequency. The images were produced using a modified spin-warp (17, 18) sequence with 7x = 300 ms and TE = 25 ms for the proton image, and 7R = 5 s and 7z = 25 ms for the fluorine19 image. The matrix size was 256 X 256 and the field of view 11 cm for both images. To increase the signal-to-noise ratio, four acquisitions for proton and eight acquisitions for fluorine19 images were averaged. The images were acquired on a General Electric 2T CSI spectrometer and processed using the 2D FT software. The concentration of

Uptake and Elimination of Isoflurane in Rabbit Tissue Studied with 19F NMR Spectroscopya

Proton nuclear magnetic resonance spectroscopy was used to evaluate changes in plasma lipoproteins in patients with squamous cell head and neck cancer. Plasma from 14 patients was analyzed by proton nuclear magnetic resonance spectroscopy, and line widths and fast and slow methyl and methylene transverse relaxation values were obtained. In addition, the lipid and lipoprotein concentrations in the sera of these patients were measured by standard biochemical techniques. Preliminary results suggested that squamous cell carcinomas of the head and neck with nodal metastases are associated with measureable changes in slow methylene transverse relaxation values, as compared with controls. These findings indicate the presence of a new lipoprotein complex in patients with squamous cell carcinomas of the head and neck with nodal metastases.