Bupropion Hydrochloride.

Bupropion hydrochloride is a norepinephrine-dopamine disinhibitor (NDDI) approved for the treatment of depression and smoking cessation. Bupropion is a trimethylated monocyclic phenylaminoketone second-generation antidepressant, which differs structurally from most antidepressants, and resides in a novel mechanistic class that has no direct action on the serotonin system. Comprehensive chemical, physical, and spectroscopic profiles are presented. This analytical profile provides an extensive spectroscopic investigation utilizing mass spectrometry, one- and two-dimensional NMR, solid-state NMR, IR, NIR, Raman, UV, and X-ray diffraction. The profile also includes significant wet chemistry studies for pH, solubility, solution, and plasma stability. Both HPLC and UPLC methodology are presented for bupropion and its related impurities or major metabolites. The profile concludes with an overview of biological properties that includes toxicity, drug metabolism, and pharmacokinetics.

Long-term stability study of Prussian blue-A quality assessment of water content and cyanide release.

CONTEXT: Prussian blue, ferric hexacyanoferrate is approved for (oral) treatment of internal contamination with radioisotopes of cesium or thallium. Cyanide makes up 35-40% of Prussian blue's molecular composition; thus, cyanide may be released during transit through the digestive tract under physiological pH conditions. OBJECTIVES: The purpose of this study is to assess the long-term stability of Prussian blue drug products and active pharmaceutical ingredients and its impact on cyanide release. The study involves the determination and comparison of the loss in water content and cyanide released from Prussian blue under pH conditions that bracket human physiological exposure. METHODS: Test samples of active pharmaceutical ingredient and drug product were stored for 10 years at ambient temperatures that mimic warehouse storage conditions. Water loss from Prussian blue was measured using thermogravimetric analysis. An in vitro physiological pH model that brackets gastric exposure and gastrointestinal transit was utilized for cyanide release. Prussian blue was incubated in situ at pH: 1.0, 5.0, and 7.0 @ 37°C for 1-24 h. Cyanide was measured using a validated colorimetric method by UV-Vis spectroscopy. RESULTS: Although the water content (quality attribute) of Prussian blue active pharmaceutical ingredient and drug product decreased by about 10.5% and 13.8%, respectively, since 2003, the cyanide release remained comparable. At pH of 7.0 for 24 h cyanide released from active pharmaceutical ingredient-1 was 21.33 ± 1.76 µg/g in 2004, and 28.45 ± 3.15 µg/g in 2013; cyanide released from drug product-1 was 21.89 ± 0.56 µg/g in 2004, and 27.31 ± 5.78 µg/g in 2013. At gastric pH of 1.0 and upper gastrointestinal pH of 5.0, the data for active pharmaceutical ingredients and drug products were also comparable in 2013. The cyanide release is still pH-dependent and follows the same trend as observed in 2003 with minimum release at pH of 5.0 and maximal release at pH of 1.0. In summary, this is the long-term stability study of Prussian blue which correlates cyanide release to water loss. Cyanide released from Prussian blue was maximum at pH of 1.0 (47.47 µg/g) and minimum at pH of 5.0-7.0 (20.01 µg/g). CONCLUSIONS: Based on maximal dose, maximal residence time in stomach and intestine, the maximal cyanide released from Prussian blue is about 1.31 mg, which is far below the minimal lethal dose of cyanide of 50 mg, and therefore does not present a safety concern following long-term storage.

Product quality of parenteral vancomycin products in the United States.

In response to concerns raised about the quality of parenteral vancomycin products, the U.S. Food and Drug Administration (FDA) is investigating the product quality of all FDA-approved parenteral vancomycin products available in the United States. Product quality was evaluated independently at two FDA Office of Testing and Research (FDA-OTR) sites. In the next phase of the investigation, being done in collaboration with the National Institute of Allergy and Infectious Diseases, the in vivo activity of these products will be evaluated in an appropriate animal model. This paper summarizes results of the FDA investigation completed thus far. One site used a validated ultrahigh-pressure liquid chromatography method (OTR-UPLC), and the second site used the high-performance liquid chromatography (HPLC) method for related substances provided in the British Pharmacopeia (BP) monograph for vancomycin intravenous infusion. Similar results were obtained by the two FDA-OTR laboratories using two different analytical methods. The products tested had 90 to 95% vancomycin B (active component of vancomycin) by the BP-HPLC method and 89 to 94% vancomycin by OTR-UPLC methods. Total impurities were 5 to 10% by BP-HPLC and 6 to 11% by OTR-UPLC methods. No single impurity was >2.0%, and the CDP-1 level was ≤ 2.0% across all products. Some variability in impurity profiles of the various products was observed. No adverse product quality issues were identified with the six U.S. vancomycin parenteral products. The quality parameters of all parenteral vancomycin products tested surpassed the United States Pharmacopeia acceptance criteria. Additional testing will characterize in vivo performance characteristics of these products.

Development and application of a validated HPLC method for the analysis of dissolution samples of levothyroxine sodium drug products.

A rapid, selective, and sensitive gradient HPLC method was developed for the analysis of dissolution samples of levothyroxine sodium tablets. Current USP methodology for levothyroxine (L-T(4)) was not adequate to resolve co-elutants from a variety of levothyroxine drug product formulations. The USP method for analyzing dissolution samples of the drug product has shown significant intra- and inter-day variability. The sources of method variability include chromatographic interferences introduced by the dissolution media and the formulation excipients. In the present work, chromatographic separation of levothyroxine was achieved on an Agilent 1100 Series HPLC with a Waters Nova-pak column (250 mm × 3.9 mm) using a 0.01 M phosphate buffer (pH 3.0)-methanol (55:45, v/v) in a gradient elution mobile phase at a flow rate of 1.0 mL/min and detection UV wavelength of 225 nm. The injection volume was 800 µL and the column temperature was maintained at 28°C. The method was validated according to USP Category I requirements. The validation characteristics included accuracy, precision, specificity, linearity, and analytical range. The standard curve was found to have a linear relationship (r(2)>0.99) over the analytical range of 0.08-0.8 µg/mL. Accuracy ranged from 90 to 110% for low quality control (QC) standards and 95 to 105% for medium and high QC standards. Precision was <2% at all QC levels. The method was found to be accurate, precise, selective, and linear for L-T(4) over the analytical range. The HPLC method was successfully applied to the analysis of dissolution samples of marketed levothyroxine sodium tablets.

Molecular weight determination for colloidal iron by Taguchi optimized validated gel permeation chromatography.

Method development of gel permeation chromatography (GPC) is a time-consuming task, since finding appropriate operating conditions has traditionally been a trial-and-error process. A novel approach in the field of GPC using experimental design called Taguchi is presented. This experimental design was used to compare the net effects of various conditions which were both qualitative and quantitative in nature. Quantitative factors included mobile phase pH, flow rate, temperature of column and detector, and injection volume. The qualitative factors were treated as noise which included enclosure of GPC system and position of waste container with respect to refractive index detector. The method was efficient as opposed to a one-factor-at-a-time approach. Taguchi optimized conditions included pH of 7.2, flow rate of 0.4 mL/min, temperature of 35 degrees C for column and detector, as well as injection volume of 10 microL. The optimized factors yielded acceptable results in terms of weight average molecular weight (m.w.), standard deviation and signal-to-noise ratio. Standard curves were constructed using dextran m.w. standards (12,000-270,000 Da) over the analytical range. The method was validated according to ICH guidelines. Log-linear function was used for m.w. standard curve and weight average m.w. was calculated utilizing trapezoidal approach. A correlation coefficient of >0.99 was obtained for both intra-day and inter-day standard calibration curves. Inter-day accuracy ranged from 91 to 108% and precision was <2.0%.

A modern view of excipient effects on bioequivalence: case study of sorbitol.

PURPOSE: To examine the effect of common excipients such as sugars (sorbitol versus sucrose) on bioequivalence between pharmaceutical formulations, using ranitidine and metoprolol as model drugs. METHODS: Two single-dose, replicated, crossover studies were first conducted in healthy volunteers (N=20 each) to compare the effect of 5 Gm of sorbitol and sucrose on bioequivalence of 150 mg ranitidine or 50 mg metoprolol in aqueous solution, followed by a single-dose, nonreplicated, crossover study (N=24) to determine the threshold of sorbitol effect on bioequivalence of 150 mg ranitidine in solution. RESULTS: Ranitidine Cmax and AUC0-infinity were decreased by approximately 50% and 45%, respectively, in the presence of sorbitol versus sucrose. Similarly, sorbitol reduced metoprolol Cmax by 23% but had no significant effect on AUC0-infinity. An appreciable subject-by-formulation interaction was found for ranitidine Cmax and AUC0-infinity, as well as metoprolol Cmax. Sorbitol decreased the systemic exposure of ranitidine in a dose-dependent manner and affected bioequivalence at a level of 1.25 Gm or greater. CONCLUSIONS: As exemplified by sorbitol, some common excipients have unexpected effect on bioavailability/bioequivalence, depending on the pharmacokinetic characteristics of the drug, as well as the type and amount of the excipient present in the formulation. More research is warranted to examine other 'common' excipients that may have unintended influence on bioavailability/bioequivalence.

Developmental neurotoxicity of ketamine: morphometric confirmation, exposure parameters, and multiple fluorescent labeling of apoptotic neurons.

Ketamine is a widely used pediatric anesthetic recently reported (C. Ikonomidou et al., 1999, Science 283, 70-74) to enhance neuronal death in neonatal rats. To confirm and extend these results, we treated four groups of PND 7 rats with seven sc doses, one every 90 min, of either saline, 10 mg/kg ketamine, 20 mg/kg ketamine, or a single dose of 20 mg/kg ketamine. The repeated doses of 20 mg/kg ketamine increased the number of silver-positive (degenerating) neurons in the dorsolateral thalamus to a degree comparable to previous results (Ikonomidou et al., 1999, Science 283, 70-74), i.e., 28-fold vs. 31-fold respectively. However, blood levels of ketamine immediately after the repeated 20 mg/kg doses were about 14 micrograms/ml, about seven-fold greater than anesthetic blood levels in humans (J. M. Malinovsky et al., 1996, Br. J. Anaesth. 77, 203-207; R. A. Mueller and R. Hunt, 1998, Pharmacol. Biochem. Behav. 60, 15-22). Levels of ketamine in blood following exposure to the multiple 10 mg/kg doses of ketamine or to a single 20 mg/kg dose ranged around 2-5 micrograms/ml; although these blood levels are close to an anesthetic level in humans, they failed to produce neurodegeneration. To investigate the mode of ketamine-induced neuronal death, coronal sections were stained with both Fluoro-Jade B (a green fluorescent stain selective for neurodegeneration) and DAPI (a blue DNA stain), as well as for caspase-3 (using an antisera labeled red with rhodamine). These histochemical results confirmed the developmental neurotoxicity of ketamine, demonstrated that Fluoro-Jade B (FJ-B), like silver methods, successfully stained degenerating neurons in neonatal rats, and indicated that ketamine acts by increasing the rate of neuronal apoptosis.

Decreased cisplatin/DNA adduct formation is associated with cisplatin resistance in human head and neck cancer cell lines.

PURPOSE: To evaluate the correlation between cisplatin sensitivity, intracellular glutathione, and platinum/DNA adduct formation (measured by atomic absorption spectroscopy) in a series of seven head and neck cancer cell lines, and to evaluate the effect of biochemical modulation of glutathione on platinum/DNA adduct formation and repair. METHODS: Cisplatin/DNA adducts were measured by atomic absorption spectroscopy. Glutathione content was measured by enzymatic assay and was modulated with buthionine sulfoximine. Apoptosis was measured by double-labeled flow cytometry. RESULTS: Intracellular glutathione concentration was strongly correlated with cisplatin resistance (P = 0.002, R2 = 0.7). There was also a statistically significant inverse correlation between cisplatin/DNA adduct formation and the IC50 for cisplatin in these cell lines. (P = 0.0004, R2 = 0.67). In addition, resistant cells were able to repair approximately 70% of cisplatin/DNA adducts at 24 h, while sensitive cells repaired less than 28% of adducts in the same period. However, despite the positive correlation between cellular glutathione and cisplatin resistance, there was no direct correlation between intracellular glutathione concentration and platinum/DNA adduct formation. Further, depletion of intracellular glutathione by buthionine sulfoximine did not dramatically alter formation of cisplatin/DNA adducts even though it resulted in marked increase in cisplatin cytotoxicity and was associated with increased apoptosis. CONCLUSIONS: These results suggest that glutathione has multiple effects not directly related to formation of cisplatin/DNA adducts, but may also be an important determinant of the cell's ability to repair cisplatin-induced DNA damage and resist apoptosis.

An evaluation of the hemizygous transgenic Tg.AC mouse for carcinogenicity testing of pharmaceuticals. I. Evidence for a confounding nonresponder phenotype.

We have completed 2 26-wk studies to evaluate the hemizygous transgenic Tg.AC mouse, which has been proposed as an alternative short term model for testing carcinogenicity. We attempted to evaluate the response to the known rodent carcinogens cyclophosphamide, phenolphthalein, and tamoxifen and to the noncarcinogen chlorpheniramine following topical application. In the first study, a weak response (2/17 animals) was observed to the positive control 12-O-tetradecanoylphorbol 13-acetate (TPA in ethanol, 1.25 micrograms), and no response was observed to cyclophosphamide, phenolphthalein, or chlorpheniramine, despite evidence for skin penetration. The second study compared 1.25 micrograms and 6.25 micrograms of TPA in ethanol and acetone solutions. Tamoxifen was also evaluated in both solvents and orally. No significant response was observed to tamoxifen by skin paint or oral routes. Over 60% of the high dose TPA-treated animals showed no (0 or 1) papilloma response, and 30% of the animals each developed more than 32 papillomas. The heterogenous response to high dose TPA may be related to variability in the responsiveness of hemizygous animals. In light of these findings, further Tg.AC studies should employ homozygous animals, and the underlying cause for heterogeneity in the tumorigenic response of Tg.AC mice should be identified and eliminated.

MRI contrast-dose relationship of manganese(III)tetra(4-sulfonatophenyl) porphyrin with human xenograft tumors in nude mice at 2.0 T.

Previously we reported that Mn(III)tetra(4-sulfonatophenyl) porphyrin, MnTPPS4, is a contrast agent which can effectively enhance tumor detection by MRI. By imaging 30 additional athymic nude mice bearing subcutaneous MCF-7 WT human breast carcinoma xenografts, we have extended dose-contrast relationships over a wide range of intraperitoneal (IP) doses ranging from 0.025 to 0.50 mmol/kg. The benefits of IP injection are higher possible doses on a volume basis and a reduction in toxicity versus IV administration. Full coronal cross-section images have been obtained on a 2-T spectrometer. Although individual tumor masses displayed different distribution patterns, reflective of their internal morphology, single doses of 0.10 mmol/kg or greater were necessary to produce a detectable effect. At a dose of 0.50 mmol/kg, marked enhancement was produced. Multiple small dosages administered over the course of several days before imaging did not produce increased enhancement. Preliminary results on the new porphyrin derivative, MnTPPS3, indicate that the ratio of the toxic dose to the effective dose may be adjustable to render this class of agents clinically useful.

Toxicity and effects of epidermal growth factor on glucose metabolism of MDA-468 human breast cancer cells.

Epidermal growth factor (EGF) has an in vitro inhibitory effect on tumor cells which exhibit a high number of EGF receptors (EGFR). Studies were performed in order to delineate the effects of EGF on glucose metabolism of MDA-468 human breast cancer cells, which have a large number of EGFR. Glucose consumption and lactate production were found to be substantially increased in MDA-468 cells following EGF exposure, while no such effects were detected in MCF-7 breast cancer cells, which have a very low number of EGFR. When glucose levels in the growth medium were increased, the toxicity of EGF was diminished. The energetic status of MDA-468 cells perfused with growth medium containing EGF was monitored by 31P magnetic resonance spectroscopy, and no signs of compromised metabolic state or viability were noted for up to 36 h. The rate of glucose transport and phosphorylation was quantitated by 13C magnetic resonance spectroscopy, utilizing [6-13C]2-deoxyglucose, and a 97% increase was found in MDA-468 cells following EGF administration. The profound effects of EGF on glucose metabolism in cells with very high numbers of EGFR and the lack of toxicity in the perfused system may indicate that the growth-inhibitory effect is confined to the in vitro cultured cells.

Effects of 2-deoxyglucose on drug-sensitive and drug-resistant human breast cancer cells: toxicity and magnetic resonance spectroscopy studies of metabolism.

The glycolytic inhibitor 2-deoxyglucose (2-DG) was tested as a potential chemotherapeutic agent for drug-resistant cancer cells. Previously it was found that Adriamycin-resistant human MCF-7 breast cancer cells (ADR) exhibit an enhanced rate of glycolysis compared to their parent wild-type (WT) cell line (R. C. Lyon et al., Cancer Res., 48: 870-877, 1987). We now describe a specific toxic effect of 2-DG on the ADR cells, which is more than 15-fold greater than for WT cells. Using 31P magnetic resonance spectroscopy of perfused MCF7 cells we continuously monitored the accumulation of 2-deoxyglucose 6-phosphate together with concomitant changes in other phosphate-containing metabolites. Kinetic measurements demonstrated that ADR cells accumulated 2-deoxyglucose 6-phosphate faster and to a greater extent than WT cells, while their depletion of high energy compounds (ATP, phosphocreatine) was more pronounced and became irreversible earlier. The phosphorylation of 2-DG could be followed more effectively by the use of 13C magnetic resonance spectroscopy of 2-DG enriched with 13C at C-6, since the signals of 2-DG and 2-deoxyglucose 6-phosphate are clearly resolved and, unlike 31P magnetic resonance spectroscopy, there are no other interfering signals. With the use of this technique with ADR and WT cells the rate of phosphorylation of 2-DG was found to be 11.2 x 10(-4) and 6.5 x 10(-4) mmol/min/mg protein, respectively. The results of these studies indicate that differences in the biochemistry of energy metabolism of resistant cells may make them targets for energy antimetabolites.

Concerning antisense inhibition of the multiple drug resistance gene.

Recently, Vasanthakumar and Ahmed reported (Vasanthakumar, G.; Ahmed, N.K., Cancer Communications 1:225-232; 1989) a complete inhibition of the multiple drug resistance gene (MDR1) in the K562/III erythroleukemia cells, using a 15 bases-long methylphosphonate oligodeoxynucleotide analog. The sequence used, however, contained three mismatches relative to the corresponding fragment of the human MDR1 gene and, hence, the results reported cannot at present be regarded as a classical antisense effect. We have made attempts to inhibit the expression of the MDR1 gene in MCF-7 human breast cancer cells selected for resistance to Adriamycin using phosphorothioate analogs of oligodeoxynucleotides. Studies with model 35S-labeled-phosphorothioates indicated poor uptake of the compounds into the cells; the radioactivity was located mainly in the soluble fraction (cytoplasm), but membranes and the nuclear fraction were also labeled. Unmodified oligodeoxynucleotides were toxic to the cells, whereas the phosphorothioates were not. The MDR1 inhibition with phosphorothioates was studied by measuring their effects on adriamycin toxicity and by immunocytochemical titration of P170. Elevation of adriamycin cytotoxicity consistent with a decreased drug resistance was observed with one antisense sequence, but the immunocytochemical assay indicated only slight inhibition of the synthesis of P170. In the wild type (drug sensitive) MCF-7 cells phosphorothioates decreased adriamycin toxicity in a sequence-independent manner. The results indicate that the effects of antisense oligodeoxynucleotides on cells are complex. Computer simulation of the secondary structure of MDR1 mRNA indicated not only extensive folding but, also, the presence of many regions not involved in intramolecular hybridization, which are of potential interest as targets for antisense oligodeoxynucleotides.

Metalloporphyrin magnetic resonance contrast agents. Feasibility of tumor-specific magnetic resonance imaging.

The criteria for tumor-specific MRI contrast agents are considered. The metalloporphyrins have been shown to be potential tumor-specific contrast agents due to their selective retention by cancer cells. The Mn(III) water-soluble porphyrin complexes are preferred on the basis of relaxivity and stability. Protein binding in human plasma enhances the relaxivity of Mn(III)-tetraphenylsulfonato-porphyrin (MnTTPS). In preliminary work this agent was shown to enhance MRI contrast at 0.5 T in subcutaneous human colon carcinomas grown in nude mice. Here we report further evidence of enhanced contrast in the same system, and extend this work to human breast tumors in nude mice using a 2 T animal imager. Questions of metalloporphyrin stability, toxicity and dose-contrast relationship are discussed.

Glucose metabolism in drug-sensitive and drug-resistant human breast cancer cells monitored by magnetic resonance spectroscopy.

Glucose utilization and lactate production have been monitored as a function of time using 13C magnetic resonance spectroscopy and [13C1]-glucose with perfused wild type MCF-7 human breast cancer cells and a drug-resistant (AdrR) cell line derived from them. Compared to wild type cells, AdrR cells exhibited an enhanced (3-fold) rate of glycolysis, indicating an increased demand for ATP production. We have investigated the effects of glucose depletion and azide, an inhibitor of oxidative phosphorylation, on the levels of intracellular phosphates (Pi, ATP) and intracellular pH using 31P magnetic resonance spectroscopy and on the rates of glycolysis. In both cell lines, ATP levels and the rates of glucose utilization and lactate production were invariant in the presence of azide. ATP production, especially in AdrR cells, was highly dependent on active glucose metabolism. The results of these direct measurements confirm that these cells survive by predominantly utilizing glycolysis. Glutamate and myo-inositol were observed in 13C spectra of acid extracts of AdrR but not wild type cells. Both metabolites are potential substrates in drug detoxification. These differences in rates of glycolysis, ATP production, and the production of certain metabolites may reflect metabolic adaptations associated with the development of drug resistance.

Phospholipid metabolism in cancer cells monitored by 31P NMR spectroscopy.

Addition of choline, ethanolamine, or hemicholinium-3 (a choline kinase inhibitor) to the perfusate of human breast cancer cells monitored by 31P NMR spectroscopy resulted in significant changes to phosphomonoester (PME) and phosphodiester (PDE) signals. These results enable us to assign the PMEs to phosphcholine (PC) and phosphoethanolamine (PE), the PDEs to glycerophosphorylcholine and glycerophosphorylethanolamine, and to define the pathways producing them. The PMEs are products of choline and ethanolamine kinases, the first steps in phospholipid synthesis; and the PDEs are substrates of glycerophosphorylcholine phosphodiesterase, the last step in phospholipid catabolism. Furthermore, PC and PE peaks are twice as intense in cells at log phase versus confluency. We also observed these signals in vivo in human colon and breast tumors grown in mice. Since PMEs are low in most nonproliferating tissues, they could form a basis for noninvasive diagnosis. Also, PE and PC are situated between the control enzymes of two major synthetic pathways and will allow noninvasive 31P NMR studies of these pathways in intact cells and in vivo.

Studies on the mechanism of selective retention of porphyrins and metalloporphyrins by cancer cells.

Comparative studies of the toxicity, stability, and retention of the water-soluble porphyrin, tetraphenylporphyrin sulfonate (TPPS), and its complex with Mn(III), have been made with the human breast cancer cell line MCF-7 wild type, and an adriamycin-resistant line derived from it, termed AdrR. Based on growth inhibition, we determined the maximum non-toxic concentration of MnTPPS tolerated by these cells. The integrity of MnTPPS in vitro was investigated by fluorescence microscopy, and we found that there is very little dissociation of MnTPPS within these cells within 4 days. We report novel proton magnetic resonance relaxation measurements of the bulk water of cells in a gel matrix undergoing perfusion. A slightly greater net uptake of MnTPPS in the wild-type cells was observed compared to AdrR; however, there was no significant difference in retention of MnTPPS. These results indicate that over a period of several hours the mechanism of selective retention of these compounds in tumour cells is not due to specific interaction with heme-binding protein, of which there is enhanced expression in the resistant cells. The fact that the net rate of washout of MnTPPS is approximately the same as the net rate of uptake also appears to eliminate compartmentalization or enzymatic modification of MnTPPS within these cells.

Tissue distribution and stability of metalloporphyrin MRI contrast agents.

Mn(III), Fe(III), and Gd(III) complexes of tetrakis(4-sulfonatophenyl)porphyrin (TPPS) and several other porphyrins were evaluated as potential MRI contrast agents. Based on consideration of relaxivity and stability properties in solution, MnTPPS was found to be the compound of choice. At pH 7 the Gd and Mn complexes significantly enhanced the water proton relaxation rate, while the relaxivity of FeTPPS exhibited a significant loss of relaxivity above pH 6 due to oxy-dimer formation. Although GdTPPS exhibited the highest relaxivity in solution, this property was rapidly lost due to dissociation of the metal ion. By contrast MnTPPS remained stable in human plasma after incubation for 9 days. Upon intravenous injection into athymic mice bearing subcutaneous human colon carcinoma xenografts, MnTPPS provided enhanced relaxation of the tissue water in several excised mouse tissues, notably kidney, liver, and tumor. The results at a fixed field (0.25 T) and relaxation dispersion studies showed decreases in water relaxation rates with time for kidney and liver, but an increase for the tumor, with a maximum near 4 days at the highest dose used.

A perfusion technique for 13C NMR studies of the metabolism of 13C-labeled substrates by mammalian cells.

We describe an improved perfusion system designed for NMR studies with 13C-labeled metabolites. Using this system we have monitored the uptake and metabolism of [13C]acetate and [13C]glucose in pooled human lymphocytes cast in an agarose gel thread.