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Low-density lipoproteins (LDLs) are a naturally occurring endogenous nanoplatform in mammalian systems. These nanoparticles (22 nm) specifically transport cholesterol to cells expressing the LDL receptor (LDLR). Several tumors overexpress LDLRs presumably to provide cholesterol to sustain a high rate of membrane synthesis. Amphiphilic gadolinium (Gd)-diethylenetriaminepentaacetic acid chelates have been incorporated into the LDL to produce a novel LDLR-targeted magnetic resonance imaging (MRI) contrast agent. The number of Gd chelates per LDL particle ranged between 150 and 496 Gd(III). In vitro studies demonstrated that Gd-labeled LDL retained a similar diameter and surface charge as the native LDL particle. In addition, Gd-labeled LDL retained selective cellular binding and uptake through LDLR-mediated endocytosis. Finally, Gd-labeled LDLs exhibited significant contrast enhancement 24 hours after administration in nude mice with human hepatoblastoma G2 xenografts. Thus, Gd-labeled LDL demonstrates potential use as a targeted MRI contrast agent for in vivo tumor detection.

Rationale and objectives Over-expression of low-density lipoprotein receptors (LDLRs) occurs in many types of malignancies and is related to the requirement for lipids for rapid proliferation of the tumors. On the other hand, LDLRs that are unable to bind LDL are found on hepatocytes of patients with familial hypercholesterolemia (FH), a genetic disease that leads to premature atherosclerosis and death. The highly selective binding of LDL to LDLR makes these particles ideal carriers of therapeutic and diagnostic contrast agents into the targeted cells. The objectives of this paper are to examine whether a prototype contrast agent (PTIR267) with dual detection properties is suitable for labeling of LDL particles for in vivo detection of LDLR by magnetic resonance imaging (MRI) and for in vitro monitoring of cellular localization by confocal fluorescence microscopy. Materials and methods PTIR267 is a lipophilic GdDTPA derivative conjugated to a fluorescent dye. The conjugated dye molecule makes the probe sufficiently water soluble to allow labeling of LDL by a brief incubation of LDL with PTIR267 dissolved in PBS at 37°C (mole ratio LDL: PTIR267 = 0.09:1). The molar relaxivity of PTIR267 in saline is 26 mM−1s−1. Specific LDLR-mediated uptake of PTIR267-labeled LDL was demonstrated in vitro by confocal fluorescence imaging of B16 melanoma cells using confocal fluorescence imaging. In vivo uptake of PTIR267-labeled LDL by a subcutaneously implanted B16 melanoma in mice leads to 30% decrease in longitudinal relaxation time (T1) in the tumor. In vivo uptake of PTIR267-labeled LDL leads to 70% decrease in T1 in a normal C57BL/6 mouse liver; however, in the liver of LDL receptor gene knockout (LDLr−/−) mice with C57BL/6 background, only 12% decrease in T1 is observed. Conclusions The dual fluorescence and MR imaging properties of PTIR267, combined with the ease of LDL labeling, suggest that it will be a useful tool for optimization of LDLR-targeted cancer diagnosis or therapy and for monitoring the efficacy of gene therapy of FH.

One of the biochemical "hallmarks" of malignancy is enhanced tumor glycolysis, which is primary due to the overexpression of glucose transporters (GLUTs) and the increased activity of mitochondria-bound hexokinase in tumors. Easy methods for assessing glucose utilization in vitro and in vivo should find widespread application in biological and biomedical studies, as illustrated by the adoption of FDG PET imaging in medicine. We have recently synthesized a new NIR fluorescent pyropheophorbide conjugate of 2-deoxyglucose (2DG), Pyro-2DG, as a GLUT-targeted photosensitizer. In this study, we have evaluated the in vivo uptake of Pyro-2DG and found that Pyro-2DG selectively accumulated in two tumor models, 9L glioma in the rat and c-MYC-induced mammary tumor in the mouse, compared to surrounding normal muscle tissues at a ratio of about 10:1. By simultaneously performing redox ratio and fluorescence imaging, a high degree of correlation between the PN/(Fp+PN) redox ratio, where PN denotes reduced pyridine nucleotides (NADH) and Fp denotes oxidized flavoproteins, and the Pyro-2DG uptake was found in both murine tumor models, indicating that Pyro-2DG could serve as an extrinsic NIR fluorescent metabolic index for the tumors. The fact that only a low level of correlation was observed between the redox ratio and the uptake of Pyro-acid (the free fluorophore without the 2-deoxyglucose moiety) supports the hypothesis that Pyro-2DG is an index of the mitochondrial status (extent of PN reduction) of a tumor.

Rationale and objectives The purpose of this study was to define and characterize carbocyanine labeled low-density lipoprotein (LDL) to be used in the optical imaging of LDL receptor (LDLr)-overexpressing tumor models. Materials and methods 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was used to label LDL (DiI-LDL). Scatchard plots were generated to determine the maximum binding capacity Bmax and dissociation constants KD of DiI-LDL in B16 melanoma (B16) and hepatoblastoma G2 (HepG2) cell lines. Selective uptake of DiI-LDL into both tumor cells and corresponding subcutaneous tumors in mice were demonstrated by confocal microscopy and three-dimensional Cryo-imaging, respectively. Results The labeling efficiency of DiI-LDL was 61 ng DiI/μg LDL protein (34 mol DiI/mol LDL protein). Bmax and KD for B16 cells were 6.311 ng LDL/mg cell protein and 60.38 μg protein/mL (117 nM), respectively. Bmax and KD were 7.573 ng LDL/mg cell protein and 26.79 μg protein/mL (52 nM) for HepG2 cells, respectively. Confocal microscopic images showed specific uptake of DiI-LDL throughout the cytoplasm in the B16/HepG2 cells. Cryo-imaging demonstrated preferential accumulations of DiI-LDL in the viable tumor regions of both B16 and HepG2 tumors compared with their adjacent normal tissues and corresponding necrotic tumor regions. In addition, uptake of DiI-LDL by the HepG2 tumor was much higher than that of the B16 tumor, consistent with the fact that the probe binding affinity for LDLrs of HepG2 cells is 2.3 times that of B16 cells. Conclusion This study suggested that carbocyanine labeled LDL could be used for optical imaging of tumors overexpressing LDLr.

Rational and Objectives. Dynamic contrast-enhanced (DCE) MRI offers the potential to provide quantitative maps of tumor perfusion parameters and is therefore expected to play an important role in the study of cancer in small animal models. Extraction of such information from DCE-MRI data requires a methodology for determination of the arterial input function (AIF) for the target tissues. An MRI based method for observation of the AIF in a mouse model is demonstrated in the present report. Materials and Methods. A series of short-axis cardiac images was acquired during the first pass of a bolus of Gadodiamide using a low-resolution, EEG-gated, saturation-recovery gradient echo imaging sequence. The AIF was then extracted from the observed signal intensity changes in the left ventricle (LV) blood pool. Results. The proposed technique provides sufficient temporal and spatial resolution to accurately characterize the AIF of Gadodiamide in mouse models. The AIF was observed in 4 mice and was found to be qualitatively similar to that previously observed in larger animals. However, significant inter-animal variability in the precise form of the AIF was evident. Conclusions. The proposed method for determination of AIF in mice has been shown to be effective and reliable. The inter-animal variability observed in the present study suggests that the AIF should be measured in each animal undergoing analysis by DCE-MRI.

Contains fulltext : 142496.pdf (Publisher’s version ) (Closed access) RATIONALE AND OBJECTIVES: Combinations of chemotherapy and fractionated radiation therapy are the currently preferred nonsurgical treatment methods for squamous cell carcinoma of the head and neck, but to the authors' knowledge there is no reliable marker for predicting therapeutic response. Early identification of nonresponders would allow prompt replacement of ineffective, toxic therapy by alternative, potentially more effective procedures. Frequent regional node involvement facilitates surface coil investigation with phosphorus-31 magnetic resonance spectroscopy. MATERIALS AND METHODS: P-31 magnetic resonance spectra were acquired from 12 patients before radiation therapy or chemotherapy. In vivo three-dimensional localized P-31 nuclear magnetic resonance chemical shift imaging was performed with a 1.5-T clinical imager and a dual-tuned H-1/P-31 surface coil. Proton decoupling and nuclear Overhauser enhancement were used to improve sensitivity and resolve overlapping signals in the phosphomonoester region of the spectrum. RESULTS: The average pretreatment ratio of phosphomonoester to beta-nucleoside triphosphate was significantly smaller in complete responders (n = 4) than in incomplete responders (partial responders plus nonresponders, n = 8) (0.0 +/- 0.0 vs 1.22 +/- 0.17 [P = .004]). CONCLUSION: Results of this preliminary study suggest that H-1-decoupled P-31 magnetic resonance spectroscopy may prove to be a useful predictor of therapeutic response in head and neck cancers.

Rationale and Objectives The authors performed this study to evaluate the selective acidification of a human melanoma xenograft in mice with severe combined immunodeficiency with the induction of hyperglycemia (mean blood glucose level ± standard error of the mean, 26 mmol/L ± 1) and the intraperitoneal administration of metaiodobenzylguanidine (MIBG, 30 mg/kg), α-cyano-4-hydroxycinnamate (CNCn, 300 mg/kg), lonidamine (100 mg/kg), cariporide (HOE642, 160 mg/kg), or 4,4′-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS, 50 mg/kg). Materials and Methods The intra- and extracellular pH levels of tumor were estimated from the chemical shifts of inorganic phosphate and 3-aminopropylphosphonate, respectively, with phosphorus-31 nuclear magnetic resonance (MR) spectroscopy. The relative level of steady-state lactate was monitored with hydrogen-1 MR spectroscopy. Results In small tumors (≤8.0 mm), hyperglycemia decreased the intra- and extracellular pH levels by less than 0.2. The combination of hyperglycemia and MIBG decreased the intra- and extracellular pH levels by approximately 0.4 and 0.6, respectively, and lowered the β-nucleoside triphosphate (NTP)/inorganic phosphate (P i ) ratio of tumor and liver by about 60% and 25%, respectively. The combination of hyperglycemia, MIBG, and CNCn produced a transient decrease in the intracellular pH of about 0.6. The combination of hyperglycemia and lonidamine produced a sustained (>3 hours) 0.8-unit decrease in intracellular pH and an 83% and 100% decrease in PCr/P i and β-NTP/P i ratios, respectively. The combination of hyperglycemia, MIBG, cariporide, and DIDS produced a gradual decrease in intra- and extracellular pH by 1.1 and 1.0, respectively. The relative level of steady-state lactate concentration in tumors increased 10% with hyperglycemia alone, about 20% with MIBG plus hyperglycemia, and increased more than twofold when hyperglycemia was combined with MIBG and CNCn administration. Conclusion These preliminary data suggest that hyperglycemia and combinations of respiratory and ion transport inhibitors can be used to selectively acidify tumors and, thereby, sensitize them to hyperthermia or other pH-sensitive therapeutic modalities.

Introduction: Meta-iodobenzylguanidine (MIBG) in its 131 I-labeled form is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors. This well established drug may have additional clinical applications as a radiosensitizer or hyperthermic agent, i.e. , MIBG reportedly inhibits mitochondrial respiration in vitro . The mechanism for MIBG inhibition of cellular oxygen consumption is uncertain. Moreover, MIBG reportedly stimulates glycolysis both in vitro and in vivo . Our studies show the effect of MIBG on 9L glioma oxygen consumption and redox status with tumors cells in vitro and in vivo . Materials and Methods: The effects on electron transfer were determined by following oxygen consumption with a Clark oxygen electrode. Fluorescence measurements were used to determine effects of MIBG on intracellular electron acceptors, NADPH and flavoproteins, in vitro and in vivo . 31 P-NMR was used to determine alterations in tumor cell pH in vivo . Results: Our results show the inhibition of oxygen utilization with MIBG for cell suspensions in vitro . The same results were demonstrated for tumor cell suspensions rapidly isolated from tumors grown in rats. Moreover, NAD(P)H and flavoprotein (Fp) fluorescence changes were observed to rapidly occur following MIBG addition in vitro . Changes in intracellular pH measured with 31 P-NMR, in vivo , precede the changes in fluorescence of NAD(P)H and Fp obtained with frozen sections of tumor. Conclusions: We conclude that 31 P-NMR measurements and fluorescence changes, following MIBG injection, can be used as criterion for selecting the proper time to treat tumors with ionizing radiation or hyperthermia.

Absolute metabolic fluxes in isolated perfused hearts have been determined by a nonlinear least squares analysis of glutamate labeling kinetics from [1-13C]glucose, [4-13C]beta-hydroxybutyrate, or [2-13C]acetate using 13C NMR spectroscopy. With glucose as substrate, the malate-aspartate shuttle flux was too slow to account for the reducing equivalents generated by glycolysis and to predict the observed oxygen consumption rate. For acetate and beta-hydroxybutyrate, the malate-aspartate shuttle had to be reversed for the network to agree with the observed oxygen consumption and glutamate labeling. Thus, an additional redox shuttle was required to reoxidize the NADH produced by cytoplasmic malate dehydrogenase. Using this model there was good agreement between the experimentally determined oxygen consumption and glutamate labeling and the calculated values of these parameters from the model for all substrates. The contribution of exogenous substrate to the overall tricarboxylic acid (TCA) cycle flux, 89.6 +/- 6.5% (mean +/- S.D.) as measured in the tissue extracts compared well with 91.4 +/- 4.2% calculated by the model. The ratio of TCA cycle flux to oxygen consumption for acetate, was 2.2 +/- 0.1, indicating that NADH production is principally accounted for by TCA cycle flux. For glucose or beta-hydroxybutyrate, this ratio was 2.9 +/- 0.2, consistent with the existence of other NADH producing reactions (e.g. glycolysis, beta-hydroxybutyrate oxidation).

A novel single-scan selective homonuclear multiple-quantum coherence-transfer technique, Sel-MQC, is presented that achieves lactate editing with complete lipid and water suppression. The method is suitable for studying tissues with high fat content and those subject to substantial motion. Frequency-selective excitation is employed to selectively prepare lactate into its multiple-quantum states; lipid and water are left in the single-quantum modes and eliminated by the multiple-quantum selection gradients. The efficiency of lipid suppression is monitored by a 2D Sel-MQC experiment which separates lipid and lactate along the multiple-quantum-evolution dimension. The spatial distribution of lactate can be imaged by the spectroscopic imaging version of Sel-MQC. Sel-MQC sequences were demonstrated both in phantoms and in vivo, using subcutaneously implanted murine EMT6 tumors.

Spontaneously hypertensive rats recieved 1 mg/kg of Adriamycin intravenously once a week for up to 12 weeks; their hearts were excised and perfused with buffer containing 4 mM [1-13C]glucose. Histological evidence of Adriamycin cardiotoxicity was evident after 8 and 12 weeks of treatment and was accompanied by a significant decrease in cardiac function. There were only minor changes in the 31P-NMR spectra in hearts following treatment; however, 13C-NMR spectra revealed decreased incorporation of label into the lactate, alanine and glutamate pools in hearts with severe tissue damage to hearts from untreated animals.

Acute adriamycin cardiotoxicity was studied in the isolated, perfused rat heart by 31P and 13C NMR spectroscopy at flow rates of 15 and 5 ml/min. Treated hearts received a total dose of 13.5 mg of adriamycin. 31P NMR spectra were collected at the beginning and end of each experiment, and cardiac function was recorded throughout. Hearts were perfused with [1-13C]glucose, and 13C NMR spectra were recorded in the presence and absence of the drug. At normal flow (15 ml/min), adriamycin caused a decline in cardiac function which was reversible when the drug was removed. There were no changes in high energy phosphate levels. The labeling of glutamate was unchanged in the presence of adriamycin; however, there was a slight increase in the labeling of lactate and alanine. At reduced flow (5 ml/min), control hearts exhibited a small decrease in ATP and phosphocreatine levels, and cardiac function was depressed. These changes were reversible when normal flow was restored. Nevertheless, adriamycin treatment at low flow caused an irreversible decline in function and in hydrolysis of ATP and phosphocreatine. At reduced flow, the control and drug-treated hearts showed similar labeling of the glutamate pool; however, there was significantly greater labeling of lactate and alanine during adriamycin treatment. These results indicate that adriamycin is more toxic under reduced flow conditions. Impairment of cardiac function by adriamycin without changes in glutamate labeling suggests that this drug alters the relationship between cardiac function and energy production.

Purpose/Objective: The cytotoxic RNase, Onconase (ONC), may show promising therapeutic implication as an alternative for cisplatin and carboplatin. ONC showed inhibition of sublethal damage and potentially lethal damage repair in addition to protein synthesis by t-RNA cleavage. This led us to consider the therapeutic effectiveness of ONC cisplatin (or carboplatin) on A549 human lung tumors, including tumors previously untreatable by either cisplatin or carboplatin.

Binding of bleomycin-A2 (Bleo-A2) to poly(dA-dT) in 100 mM sodium phosphate (pH 6.8) in D2O has been investigated by 1H NMR spectroscopy at 270 and 360 MHz. Significant spectral perturbations were observed only when the nucleic acid was in the duplex state. Of the Bleo-A2 resonances, the two bithiazole peaks exhibited the largest spectral shiffs and line broadening effects. The high field shift of these resonances was very small near room temperature and reached a maximum of about 0.27 ppm just below the thermal denaturation temperature of the nucleic acid (Tm = 60 ± 1°C). The temperature dependence of spectral perturbations may be accounted for by the formation of at least two types of Bleo-A2 complexes with the polynucleotide. Other perturbed resonances of bleomycin are the S-C H 3 and S-C H 2 of the terminal amine, the C H 2-N resonance of the bithiazole residue, and the C H (CH3)CO of the methylvaleric acid residue. The significantly perturbed resonances of the nucleic acid originate from the A(H-2), A(H-8), T(H-6) and one of the H-2′ hydrogens. Binding of the C-terminal tripeptide fragment of Bleo-A2 to poly(dA-dT) is accompanied by selective broadening of the bithiazole group. These experiments have identified potential loci of interaction on the Bleo-A2 and poly(dA-dT) molecules.

1 H NMR double resonance studies of valinomycin in (CD 3 ) 2 SO were conducted at 90 MHz (FT-mode) and 250 MHz (correlation mode) to determine the mechanism of intramolecular nuclear Overhauser effects (NOE). These studies set specific constraints on any model for the conformation of valinomycin in (CD 3 ) 2 SO and illustrate that NOE experiments of this type hold considerable promise for conformational studies of peptides, proteins and other biomolecules. The NOE's are positive at the lower frequency and negative at the higher frequency. Consideration of the theoretical dependence of the NOE on the proton-proton internuclear correlation time and on the resonance frequency indicates that these results are explained by a predominantly dipolar relaxation mechanism.

Summary The extent of segmental motion of the backbone and sidechain carbons of the glycopeptide antibiotic, bleomycin-A 2 in aqueous solution, has been determined from the natural abundance carbon-13 spin-lattice relaxation times (T 1 ) measured at 67.89 MHz. The backbone carbons of the central residues have an average T 1 of 163 msec, corresponding to an isotropic rotational correlation time of 3.9×10 −10 sec. The backbone and sidechain carbons exhibit varying degrees of motional freedom. The solution conformation of bleomycin-A 2 is discussed in terms of the conformational flexibility available to these carbons.

NH4+ and ND4+ solubilized in the cationic lyotropic mesophase have been studied by 14N, 2H, and 1H NMR. The observed dipole-dipole and quadrupole splittings of the appropriate nuclei have been interpreted by the distortion model recently proposed by Bailey and co-workers (J. Magn. Resonance 18, 344 (1975)) . From the ratios of the dipole-dipole and quadrupole splittings, the nitrogen-14 quadrupole coupling constant along the bond axis has been estimated to be 3.1 ± 0.3 MHz.

The interactions of gallium (Ga) with the ligands, EDTA, NTA, phosphate, lactate, MOPS, TRIS, and HEPES are investigated using both /sup 71/Ga and 1H nmr measurements. Both EDTA and NTA form strong complexes with gallium, which have a 1:1 stoichiometry. In alkaline solution the tetrahedral Ga(OD)/sub 4/- competes strongly with EDTA in complex formation. In the lactate complex, there are probably three lactates per gallium present. The phosphate complexes of gallium are difficult to characterize on the basis of this investigation. The buffers, MOPS, TRIS, and HEPES, do not interact with gallium significantly. The ability of the ligands to bind gallium correlates well with their ability to inhibit gallium incorporation by L1210 leukemic cells.

Attention has recently been focused on the use of the trivalent lanthanides ions as probes in NMR studies of macromolecules. Previous analyses of the shifts induced by these paramagnetic ions have assumed that the magnetic susceptibility tensor of the metals is axially symmetric. This study has tested the validity of this assumption by analyzing reported NMR shift and relaxation data for various lanthanide complexes with lysozyme. For the Gd 3+ -lysozyme complex the broadening data, fit to a ( 1 r 6 ) model, places the metal ion in close proximity to the reported x-ray position. Contrary to current assumptions, the shifts induced by Nd 3+ and Ce 3+ exhibit considerable non-axial character. On the basis of a statistical hypothesis test, the axially symmetric model was rejected with more than 97% confidence.

Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-1) tumors grown in mice have been analyzed by multinuclear NMR spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the 31 P resonances observed in vivo to specific phosphorus-containing metabolites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the 31 P-NMR spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. HPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The 1 H and 13 C resonance assignments of the majority of metabolites present in RIF-1 extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vivo and in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.

A probe, suitable for any wide-bore NMR spectrometer, was constructed for monitoring high-resolution spectra of in vivo subcutaneously implanted tumors in mice. Preliminary studies of a variety of murine tumors (MOPC 104E myeloma, Dunn osteosarcoma, colon-26, ovarian M5, and mammary adenocarcinoma as well as human colon, mammary, and lung tumors in athymic mice) indicate that the 31P NMR spectrum is a sensitive monitor of progressive metabolic changes that occur during untreated tumor growth and an early indicator of tumor response to chemotherapy, hyperthermia, and X radiation. Response to each of these therapeutic modalities is accompanied by distinctly different spectral changes.

The effects of pH, temperature and guanidine hydrochloride concentration on the structure of ubiquitin, a polypeptide which can activate adenylate cyclase and can mimic thymopoietin induced differentiation of prothymocytes, were monitored using nuclear magnetic resonance spectroscopy. This relatively small polypeptide (molecular weight of 8541) exhibits a remarkable stability towards pH and temperature changes. At 7 M guanidine hydrochloride concentration, the structure of ubiquitin is essentially a random coil.

The rate of hen egg-white lysozyme (mucopeptide N-acetylmuramoylhydrolase, EC 3.2.1.17), catalysis was determined in the presence of various metal ions (Co2+, Zn2+ and eight of the trivalent lanthanide ions). In the assay system employed, the lanthanides were found to inhibit more strongly than either Zn2+ or Co2+. The inhibition data was fitted to several models of the interactions of the metal ion with the enzyme. These models ranged in complexity from a single inhibitory metal binding site on the enzyme (two-parameter fit) to the presence of two non-independent and non-equivalent inhibitory metal binding sites (five-parameter fit). The more complicated models did not fit the data more precisely than the simplest one-site model, suggesting that the adoption of the simpler model is warranted. The fact that the association constants obtained from the simplest analysis for Co2+ (1.3 +/- 1.9 . 10(2) M-1) and Gd3+ (7.0 +/- 2.6 . 10(3) M-1) are consistent with literature values determined from spectroscopic measurements further supports the validity of the simplest model.

Development of dose dependent chronic irreversible cardiotoxicity is a key problem encountered in chemotherapy with adriamycin. Here it has been demonstrated that infusion of this agent produced distinct and largely irreversible changes in levels of phosphate metabolites and substantial acidosis that are detected by 31P NMR of the Langendorf perfused heart. Administration of the antioxidant, butylated hydroxytoluene minimizes these spectral changes but does not substantially diminish the antineoplastic activity of adriamycin. Bisantrene (CL 216,942), a noncardiotoxic anthracene with antineoplastic activity, produces only minor perturbations of the 31P spectrum of the perfused rat heart. These studies demonstrate the potential utility of employing 31P NMR to monitor acute or chronic cardiotoxicity in the perfused rat heart and for developing noninvasive in vivo NMR techniques for monitoring cardiotoxicity in experimental animals and humans.

Resonances of the indole N H protons for five of the six tryptophan residues of hen egg white lysozyme are resolved when the PMR spectrum is observed in H2O. Assignments of the resonances to specific tryptophan residues were accomplished by differential deuterium exchange rates, inhibitor perturbation and chemical modification. Indole N H resonances from all five tryptophan residues of human lysozyme also are observable.

31P NMR spectroscopy with surface coils has been used to monitor, in vivo, the phosphate metabolism of subcutaneously implanted mammary 16/C adenocarcinoma in C3H/He mice. This model tumor was studied during untreated tumor growth and after treatment with adriamycin, hyperthermia, and x-radiation. The mammary 16/C tumor exhibited a Gompertzian growth pattern. Levels of high-energy phosphate metabolites—phosphocreatine and ATP—decreased with increases in tumor mass. There was a concomitant increase in the level of Pi and a decrease in the apparent pH of the tumor. These spectral changes appear to reflect changes in tumor vascularization that accompany tumor growth, the tumor becoming progressively more hypoxic. Partial response of this tumor to chemotherapy with adriamycin was reflected in a small but measurable increase in the phosphocreatine resonance, a decrease in Pi, and a return of the intra-tumor pH to neutral. Hyperthermia resulted in progressive conversion of the 31P NMR spectrum to that of a dead tumor (high levels of Pi, small levels of residual sugar phosphates and pyridine dinucleotides, and acidic pH). X-irradiation (14.0 Gy) led to disappearance of the phosphocreatine peak within 15 min of treatment. Subsequently, this resonance grew back beyond its pretreatment level. As the tumor receded, its spectrum reflected the characteristics of aerobically metabolizing tissue (high levels of phosphocreatine and ATP and low levels of Pi and sugar phosphates).