Cellular delivery of MRI contrast agents.

Magnetic resonance imaging (MRI) is a powerful tool for acquiring images of opaque living animals with the benefit of tracking events over extended periods of time on the same specimen. Contrast agents are used to enhance regions, tissues, and cells that are magnetically similar but histologically distinct. A principal barrier to the development of MRI contrast agents for investigating biological questions is the delivery of agents across cellular membranes. Here, we describe the synthesis and in vitro testing of Gd(III)-based MRI contrast agents containing varying length polyarginine oligomers capable of permeating cell membranes. We examine the effect of the length of oligomer on T(1) enhancement and cellular uptake. Furthermore, the effect of incubation time, concentration, and cell type on uptake is explored. Toxicity and washout studies are performed in addition to MRI phantom studies.

Impaired eyeblink conditioning and decreased hippocampal volume in PDAPP V717F mice.

We examined heterozygous transgenic (Tg) mice that overexpress V717F amyloid precursor protein (APP) for delay eyeblink conditioning (EBC) and hippocampal volume with magnetic resonance imaging (MRI). Platelet-derived APP mice were significantly impaired on EBC relative to wild type (WT) litter-mate controls. T2-weighted spin echo images (62.5 x 125 x 500 microm) of the same mice were acquired under anesthesia using a 9.4T magnet. Tg mice had hippocampal to brain volume ratios that were significantly smaller than WT controls (31% smaller in the rostral dorsal hippocampus, 13-22% smaller among equal dorsal-ventral thirds of a caudal section). These results indicate that overexpression of APP or beta amyloid profoundly affects learning and memory and hippocampal volume. The results also indicate that eyeblink conditioning and quantitative MRI in mice may be useful assays to follow the progression of disease-related changes, and to test the effectiveness of potential therapeutics against Alzheimer's disease.

Monitoring of brain water by chemical shift imaging during ammonia-induced brain swelling in rats after portacaval anastomosis.

Brain edema is a leading cause of death in acute liver failure (ALF). In experimental models of ALF, an increase in the content of brain water has been inferred indirectly by measuring intracranial pressure or determined directly via analysis of brain tissue postmortem. In this study, noninvasive proton two-dimensional chemical shift imaging (2-D CSI) was used to follow the time course of the development of brain edema in a well characterized model, namely ammonium acetate infusion into rats 48 to 72 h after portacaval anastomosis (PCA). Clear differences between control and experimental rat brains were observed, with an increase of brain water signal only in the parietal cortex of the PCA + ammonia group. Selective swelling of the cerebral cortex points to a cytotoxic mechanism in the evolution of brain edema in this model. CSI signal enhancement was much greater than the gravimetrically determined water content increase. The significantly greater signal change observed with 2-D CSI may reflect enhanced proton density that results from increased water content as well as edema-related alterations in water relaxation times.

Characterization of the cerebral distribution of general anesthetics in vivo by two-dimensional 19F chemical shift imaging.

The distribution of two fluorinated anesthetics, halothane and isoflurane, in rabbit brain was mapped during the course of their uptake using 19F NMR chemical shift imaging techniques. Use of a short phase-encoding time and free induction decay acquisition minimized T2-related signal losses in two-dimensional chemical shift imaging. Localization of the brain and exclusion of surrounding tissues was achieved by the use of a surface coil. The spatial distribution of halothane and isoflurane in the brain was nonuniform and both anesthetics were detected predominantly in the cerebral cortex. During the early stages of uptake, anesthetic concentration in the brain was extremely low, but it continued to increase for several hours with sustained anesthetic delivery. Time-dependent variations in anesthetic concentration were observed in different brain regions.

In vivo 19F-NMR spectroscopic study of halothane uptake in rabbit brain.

Uptake of a fluorinated anesthetic, halothane, in rabbit brain and blood was studied using 19F-NMR spectroscopic techniques. Localized one-dimensional chemical shift imaging and non-localized one-pulse sequence were used to measure brain uptake kinetics in vivo. Halothane signal was found predominantly in the cerebral cortex. Uptake in the brain followed a first-order biexponential kinetics. The average half-lives were 4 min and 70 min, respectively, for the 'fast' and 'slow' phases of the uptake. Uptake in the arterial blood was also biexponential. However, equilibration of halothane in the brain considerably lagged behind that in arterial blood. This delay was ascribed to a 'restricted diffusion' of the anesthetic molecule into brain tissue.

In vivo 19F one-dimensional chemical shift imaging study of isoflurane uptake in rabbit brain.

19F one-dimensional chemical shift imaging and one-pulse techniques were used to follow the uptake of isoflurane in rabbit brain in vivo. Brain isoflurane appeared mainly in two adjacent slices of cerebral cortex. Both slices showed increase in anesthetic content at similar rates. A biphasic first-order uptake pattern with an initial fast component and a slower rate for the duration of the uptake was observed by the one-pulse technique. Only the slower kinetic was followed by chemical shift imaging due to constraints on temporal resolution. Anesthetic levels in arterial blood reached equilibrium significantly earlier than in brain, suggesting a 'restricted' diffusion into brain tissue for this agent.

Proton magnetic resonance spectroscopy of the brain in schizophrenic and affective patients.

Water-suppressed 1H magnetic resonance spectra were recorded from two brain regions of psychiatric patients and normal volunteers. The two regions studied were (a) the basal ganglia structures surrounding the anterior horn of the lateral ventricle and (b) the occipital cortex. N-Acetylaspartate (NAA), phosphocreatine-creatine (PCr-Cr), choline and inositol resonances were seen in both regions. Ratios of metabolite peak integrals to PCr-Cr peak integral were calculated for each spectrum. To control for partial volume effects, comparisons between patients and controls were made only from identical regions i.e. basal ganglia vs basal ganglia, and likewise for occipital cortex. Metabolite ratios from the occipital region of patients were similar to those from the occipital region of normal subjects. Bipolar patients being treated with lithium had elevated NAA/PCr-Cr in the basal ganglia region when compared to normals. These patients also demonstrated elevated choline/PCr-Cr and inositol/PCr-Cr ratios in the basal ganglia region.

Human leg neuromuscular diseases: P-31 MR spectroscopy.

Phosphorus-31 magnetic resonance (MR) spectra of leg muscles in patients with the neuromuscular diseases Duchenne dystrophy, myotonic dystrophy, postpoliomyelitis, Werdnig-Hoffmann disease, and pedal dystonia were recorded. Ratios of beta-adenosine triphosphate (ATP), inorganic phosphate (Pi), alpha-glycerophosphorylcholine (GPC), and phosphomonoesters to phosphocreatine (PCr) were calculated from peak integrals and compared with normal muscle ratios. In all diseases studied, beta-ATP/PCr and Pi/PCr values showed an increase from normal values. The extent of increase in beta-ATP/PCr was related to the clinical severity of the disease, suggesting that this could be a useful noninvasive means of monitoring effectiveness of therapy for neuromuscular disorders. In myotonic dystrophy and Werdnig-Hoffmann disease, GPC/PCr values increased greatly. The intracellular pH in Duchenne and postpoliomyelitis muscles was slightly elevated compared with that in normal muscles. Hydrogen-1 MR images of muscles showed fat infiltration in all patients, more in weaker muscles and less in stronger muscles.

Volume-selective water-suppressed proton spectra of human brain and muscle in vivo.

Volume-selective water-suppressed proton spectra were recorded from live human brain and muscle at 1.5T by combining a stimulated echo acquisition mode pulse sequence for localization and two saturation pulses for water suppression (Frahm et al., SMRM Abstracts, 1987). Metabolite signals were observed in voxels of size 4-64 cm3. Signals from -CH3 and beta-CH2 of N-acetylaspartate, =N-CH3 and =N-CH2 of phosphocreatine/creatine, -N(CH3)3 of choline and inositol protons were visible in the brain spectra from normal subjects. Differences in metabolite levels were observed between gray and white matters of brain from their water-suppressed spectra. Peaks from =N-CH3 of phosphocreatine/creatine and -N(CH3)3 of choline and carnitine were present in normal muscle spectra along with several resonances from fatty acyl chains.

Quantitative and qualitative fat analysis in human leg muscle of neuromuscular diseases by 1H MR spectroscopy in vivo.

1H MR spectra were recorded from human gastrocnemius muscle at 63.86 MHz using the body coil of the Signa scanner as transmitter and a 3-in. surface coil as receiver. The fat content of the muscle was quantified relative to that of water in a selected volume or slice. The fat/water ratio was 0.05-0.07 for normal muscle but increased to 0.5-6.0 in primary and secondary muscular disorders such as Duchenne and myotonic dystrophy, Charcot-Marie-Tooth and polio muscular atrophy, cerebral palsy, and spina bifida. In Werdnig-Hoffmann spinal atrophy the ratio was above 10. Water-suppressed and slice-selective 1H spectroscopy was used for qualitative analysis of fat. The 1H profile of gastrocnemius muscles between healthy individuals and patients with neuromuscular diseases showed two major differences. In the normal muscle spectra, the resonance from the -(CH2)n- protons at 1.6 ppm was the most pronounced, whereas in the diseased muscle spectra resonances also appeared between 1.1 and 1.4 ppm. Some diseased muscle spectra showed multiple resonances from -CH = CH- in polyunsaturated fatty acids between 5.5 and 7.0 ppm. The corresponding resonances from = CH-CH2-, 1.9-2.0 ppm, and = CH-CH2-CH =, 2.7-2.9 ppm, were also seen. These peaks are usually not detected in normal muscle.

In vivo H-1 spectroscopy in humans at 1.5 T.

Water-suppressed and section-selective proton (hydrogen-1) magnetic resonance (MR) spectra were recorded from human brain, leg muscle, liver, and heart with a 1.5-T imager. Signal from water was well suppressed, and resonances from several metabolites were consequently seen. The spectra from brains of healthy volunteers (n = 5) showed resonances from N-acetylaspartate, glutamine, aspartate, phosphocreatine/creatine, choline, taurine, and glycine. In five large brain meningiomas, resonances from N-acetyl-aspartate and phosphocreatine/creatine were either not visible or markedly decreased in intensity. The spectra from leg muscles of healthy volunteers showed resonances from protons in saturated fatty acyl chains, whereas resonances from unsaturated fatty acyl chains predominated in spectra from leg muscles of two patients with spina bifida. The spectra from livers of three healthy volunteers showed resonances from aliphatic and aromatic amino acids, choline, carnitine, and both saturated and unsaturated fatty acyl chains, and spectra from hearts of six healthy volunteers showed major resonances from phosphocreatine/creatine and taurine and smaller resonances from amino acids and fatty acyl chains.

Quantitation of phosphate metabolites in human leg in vivo.

Slice-selective 31P MR spectra were recorded from human legs with a pulse program which has only a 1.5-ms delay between excitation and acquisition. The volume of the slice was calculated from its thickness and cross-sectional area measured with the aid of a computer program. A phosphate reference standard spectrum was recorded from the same interplanar location as that of the human leg slice. Since signal integral is proportional to the phosphate concentration and to the volume of the slice, metabolite concentrations in the selected slice could be quantitated.

Estimation of tissue phospholipids by natural abundance 13C-NMR.

The total phospholipid content of excised rat muscle, liver, brain and kidney and of human muscle biopsies was estimated by natural abundance 13C-NMR after complete solubilization of the tissue membranes with excess halothane. An external dioxane capillary, calibrated against pure palmitic acid and phospholipid vesicles with known phosphate concentration, was inserted into the tissues, and the repeating methylene carbon peak area in the spectra of the halothane-treated tissues was integrated versus the dioxane reference peak area. The amount of tissue used for NMR analysis was quantitated by dry weight determination after 13C spectroscopy was completed. The phospholipid content estimated by the indirect NMR method was in good agreement with that measured by direct phosphate analysis and with literature data. For human muscle biopsies, the NMR method can also estimate the fraction of the total phospholipids which are mobile without treating the muscles with halothane. In this respect human muscles could be separated into three different groups: normal and nonspecific muscle diseases, myotonia and myopathy, Duchenne dystrophy; with increasing fraction of the mobile phospholipids in this order.

High resolution proton magnetic resonance spectroscopy of human brain and liver.

Water-suppressed and slice-selective proton spectra of live human brain exhibited several resonances that were tentatively assigned to metabolites such as N-acetylaspartate, glutamate, phosphocreatine and creatine, choline derivatives, and taurine. In the liver spectrum of a healthy volunteer, the major resonance was tentatively assigned to a fatty acyl methylene and the minor resonances to protons in carnitine, taurine, glutamate, and glutamine. In the spectrum of a cancerous liver, resonances in addition to those present in the normal liver were seen. Protein degradation in the liver with cancer was indicated by resonances from urea and from the ring protons in tryptophan, tyrosine, and phenylalanine. Furthermore, increased nucleic acid synthesis was indicated by resonances from nucleotide protons.

Metal ion binding properties of hen ovalbumin and S-ovalbumin: characterization of the metal ion binding site by 31P NMR and water proton relaxation rate enhancements.

In this study, water proton relaxation rate (PRR) enhancements have been used to characterize the binding of metal ions to native ovalbumin, ovalbumin in which phosphate has been enzymatically cleaved from one or both of the two protein phosphoserines, and a heat-stabilized form of the protein (S-ovalbumin). With Scatchard plots constructed from water PRR enhancements, it was found that native ovalbumin and S-ovalbumin had one strong binding site for Mn2+ ion (KD approximately equal to 6.0 X 10(-4) M). Alkaline phosphatase treated ovalbumin, a protein having a single phosphoserine, had one Mn2+ binding site of slightly weaker affinity (KD approximately equal to 8.3 X 10(-4) M), while acid phosphatase treated ovalbumin, a dephosphorylated protein, had two much weaker Mn2+ ion binding sites (KD approximately equal to 1.3 X 10(-3) M). Competitive binding studies on the native protein suggested that Zn2+ ion competes with Mn2+ for the single strong-affinity site (KD approximately equal to 6.1 X 10(-3) M) while Mg2+ and Ca2+ do not. In a second set of experiments, the paramagnetic contribution to the 31P spin-lattice (T1P) and spin-spin (T2P) relaxation times at three separate magnetic field strengths was measured. Correlation times tau c characterizing Mn2+-31P dipolar relaxation were estimated from the ratios of T1P/T2P at a single field and from the ratios of spin-lattice relaxation rates at three different field strengths. The correlation times so obtained, ranging from about 0.7 to 7.7 ns at the three field strengths, were used in calculating distances from the bound Mn2+ ion to the phosphoserines of native ovalbumin, S-ovalbumin, and alkaline phosphatase treated ovalbumins. It was determined that the phosphate of phosphoserine-68 was 5.95 +/- 0.26 and 6.29 +/- 0.18 A from the Mn2+ in the native and alkaline phosphatase treated protein, respectively, and 6.99 +/- 0.30 A away from the Mn2+ in S-ovalbumin. The phosphate of phosphoserine-344 was determined to be 5.31 +/- 0.20 and 5.75 +/- 0.10 A from the Mn2+ ion in native ovalbumin and S-ovalbumin, respectively. The 13C nucleus of [1-13C]galactose enzymatically transferred to the nonreducing end of the ovalbumin oligosaccharide chain was not found to be significantly relaxed by Mn2+ bound to the protein, even at 1:1 stoichiometric ratio of metal:protein. Using this, we estimate the nonreducing terminal of the ovalbumin oligosaccharide to be at least 39 A from the metal ion binding site on the protein.

A simple procedure for NMR measurements of intra- and extracellular sodium in intact tissues.

The total Na+ and both the intra- and extracellular Na+ content of excised rat and frog tissues was quantitated by 23Na NMR at 95.51 MHz. An external capillary containing 33 mM Na7[Dy(P3O10)2], resonating about 30 ppm upfield relative to the 0.00 ppm of the intracellular Na+, was inserted into the tissues. The capillary was calibrated against a concentration range of pure NaCl solution, for measurement of intracellular Na+, and against the same concentrations of NaCl solutions containing 4-6 mM K7[Dy(P3O10)2] in 50 mM histidine. Cl and 100 mM choline. Cl, for measurement of extracellular Na+. Spectra were recorded on tissues first in the absence of the shift reagent for determination of the total Na+. After addition of a K7[Dy(P3O10)2] solution to the sample, the 23Na spectra were recorded immediately so that data accumulation was completed within 15 min. Under these conditions, the extracellular Na+ resonated from 10 to 20 ppm upfield relative to the intracellular Na+, and no loss in the intensity of the extracellular Na+ resonance occurred due to the lability of dysprosium(III)tripolyphosphate (cf. Matwiyoff et al., Magn. Reson. Med. 3: 164, 1986). The intra- and extracellular Na+ content of the tissue was calculated from the integrated areas of the respective Na+ resonances and that of the calibrated capillary, from the known weight of the tissue, and from the known volumes of the solutions added.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional proton magnetic resonance of human muscle extracts.

In continuation of our previous work on one-dimensional (1D) proton nuclear magnetic resonance (1H-NMR) of normal and diseased human muscle extracts we recorded the two-dimensional (2D) J-correlated proton magnetic resonance spectra of these extracts. Significant differences between normal and diseased muscle extracts, not observed in the 1D 1H-NMR spectra, were seen from their 2D connectivity contour patterns. Taurine was not present in cerebral palsy muscle extract while both normal and scoliosis muscles contained this metabolite. Only the normal muscle had carnitine. Carnosine was present in all muscles. alpha-Ketoglutarate was found only in the diseased muscle extracts. While the amino acids lysine, cysteine and glutamine were common to normal and diseased muscles, threonine was seen only in the diseased muscles. Additional small differences were detected in the 2D patterns of human muscle extracts.

Carbon monoxide binding kinetics in "capped" porphyrin compounds.

The rate constants for CO binding to the five-coordinate ferrous iron complexes of 5,10,15,20-[pyromellitoyl(tetrakis-o-oxyoxyphenyl)]porphyrin and 5,10,15,20-[pyromellitoyl(tetrakis-o-oxypropoxyphenyl)]porphyrin have been measured and compared with the corresponding rate constants for other hemes and hemoproteins. The second-order rate constant is independent of cap size and is comparable to that of high-affinity state hemoglobin (k5 approximately 4 X 10(6) M-1s-1). Therefore, these capped porphyrins provide no steric hindrance to CO binding. In addition, a kinetic scheme involving an unusual seven-coordinate porphyrin species is described.

31p nuclear relaxation studies of para- and diamagnetic cobalamins in their two isomeric forms.

A new, naturally occurring isomeric form of vitamin B-12 was reported by us earlier (Katada, M., Tyagi, S., Nath, A., Petersen, R.L. and Gupta, R.K. (1979) Biochim. Biophys. Acta 584, 149-163). The Mössbauer parameters of various derivatives of vitamin B-12, including the one-electron reduced species, cob(II)-alamin (vitamin B-12r), exhibit differences for the two isomeric forms. This and some other observations from our laboratory indicated that the new form (vitamin B-12') presumably constitutes a conformational isomer possessing a different puckering of the corrin ring. The 31P nuclear relaxation studies of vitamins B-12r and B-12'r, B-12 and B-12', and dicyanocobalamin as compared to dicyanocobalamin' reported here are consistent with our earlier conjecture. The relaxation of the 31P nucleus in cob(II)alamins is primarily due to its dipolar interaction with the paramagnetic cobalt and therefore the measurement of the 31P nuclear relaxation times (T1) in the two isomeric forms permits us to calculate Co(II)-31P distances. The Co(II)-31P distance in vitamin B-12'r is found to be larger than that in vitamin B-12r. The 31P T1 value in the diamagnetic cob(III)-alamins is determined predominantly by its dipolar interaction with the two closest protons flanking the phosphate group, one being situated on the ribose moiety and the other on the amino propanol group. The nuclear relaxation times T1 of cyanocobalamin and dicyanocobalamin differ from ;those of their corresponding conformational isomers. The observed differences in 31P relaxation rates of the para- and diamagnetic cobalamins in their two isomeric forms can be understood on the basis of structural changes arising from variations in the nature of puckering of the corrin ring. The 31P chemical shifts in the two isomers remain essentially unchanged, indicating that the structural differences in the isomeric forms do not alter the electronic environment of the phosphorus nucleus.