William T. Evanochko

NMR study of in vivo RIF-1 tumors. Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances

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 31P 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 31P-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 1H and 13C 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 vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.

31P NMR spectroscopy of in vivo tumors

Abstract 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.

Human tumors as examined by in vivo 31P NMR in athymic mice

Abstract Surface coils have been employed to monitor in vivo 31P NMR spectra of human breast (MX-1), lung (LX-1) and colon (CX-1) tumors subcutaneously implanted in athymic mice. Spectra of these NCI screening tumors were measured during various stages of untreated growth. A progressive decrease in phosphocreatine and ATP and an increase in inorganic phosphate were observed for the MX-1 tumor line. The lung and colon tumors also exhibited a growth associated increase in sugar phosphates. The spectral characteristics of these tumors were very similar to those of corresponding murine tumors: mammary 16 C adenocarcinoma, Lewis lung sarcoma and colon 26. These data provide a basis for development of NMR techniques for monitoring human tumors in situ or in athymic mice.

1H NMR Spectroscopic Imaging of Myocardial Triglycerides in Excised Dog Hearts Subjected to 24 Hours of Coronary Occlusion

BACKGROUND Myocardial ischemic insult causes depression of fatty-acid beta-oxidation and increased fatty-acid esterification with triglyceride (TG) accumulation. This accumulation has been demonstrated to occur in the territory with diminished blood flow surrounding an infarct, ie, the region at risk. To evaluate whether the extent of TG accumulation in the canine heart after 24 hours of ischemia could be detected, we applied myocardial 1H nuclear magnetic resonance (NMR) spectroscopic imaging (SI). METHODS AND RESULTS Seven adult mongrel dogs underwent 24 hours of left anterior descending coronary artery occlusion. Postmortem, the hearts were excised and the size and location of the infarct were determined. With a Philips 1.5-T clinical NMR imaging/spectroscopic system, two-dimensional (2D) 1H NMR SI was performed. TG 1H NMR chemical shift images were reconstructed from the frequency domain spectra by numerical integration. A statistically significant (P < .05) increase in TG signal intensity was demonstrated in the region at risk compared with the nonischemic control region. There was an intermediate quantity of TG in the infarct region. Biochemical determination of tissue TG content (milligrams per gram wet weight) in the control, at-risk, and infarct regions confirmed the 1H NMR measurements. Histological evaluation with oil red O staining also demonstrated graded TG accumulation in myocytes. The highest TG levels were found in the at-risk region and the lowest levels in the control region. CONCLUSIONS By use of 2D 1H NMR SI, the present study confirms and extends previous work that demonstrates preferential accumulation of TG in the reversibly injured myocardium after 24 hours of coronary occlusion. This study provides an important step toward the clinical application of TG imaging. When TG imaging is ultimately possible, resultant data would have diagnostic, prognostic, and therapeutic implications.

Monitoring the bioenergetics of cardiac allograft rejection using in vivo P-31 nuclear magnetic resonance spectroscopy

Monitoring human cardiac allograft rejection is currently accomplished by endomyocardial biopsy. Available noninvasive methods for identifying rejection have lacked the necessary sensitivity or specificity, or both, for routine clinical application. In vivo phosphorus-31 (P-31) nuclear magnetic resonance (NMR) spectroscopy has been used for monitoring phosphorus metabolism in both animal models and humans. In the present study this technique was employed as a noninvasive means to assess the bioenergetic processes that occur during cardiac allograft rejection in a rat model. Brown Norway rat hearts were transplanted subcutaneously into the anterior region of the neck of Lewis rat recipients (allografts). Control isografts employed Lewis donors and recipients. Phosphocreatine to inorganic phosphate (PCr/Pi), phosphocreatine to beta-adenosine triphosphate (PCr/ATP beta), beta-adenosine triphosphate to inorganic phosphate (ATP beta/Pi) ratios and pH of the transplanted hearts were monitored using surface coil P-31 NMR spectroscopy (at 4.7 tesla) daily for 7 days. To allow recovery from the compromise induced by the surgical procedure, the measurements obtained on day 2 were taken as a baseline. PCr/Pi was unchanged or increased in the isografts but decreased continually in allografts, with the difference becoming significant by day 4 when compared with levels in day 2 allografts (p less than 0.005) and by day 3 when compared with levels in the isograft group (p less than 0.05). PCr/ATP beta in isografts did not change throughout the study; however, allografts demonstrated a significant decrease as early as day 3 (p less than 0.01), although a significant difference between isografts and allografts did not become manifest until day 4 (p less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of increased myocardial lipid with postischemic dysfunction (“Myocardial Stunning”) by proton nuclear magnetic resonance spectroscopy☆☆☆

Histopathologic studies have demonstrated accumulation of lipid droplets in myocardium subjected to greater than or equal to 6 h of ischemic insult. Proton nuclear magnetic resonance (NMR) spectroscopy can provide a noninvasive means to evaluate changes in tissue lipid and, potentially, to characterize the ischemic insult. To determine whether lipids accumulate with a brief ischemic insult, myocardial lipid content was evaluated by 1H NMR spectroscopy of ex-vivo samples from seven dogs in a model of postischemic dysfunction created by 15 min of left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Regional myocardial function was assessed by measuring segment length shortening with use of a pair of ultrasonic crystals placed in the ischemic zone and in the control zone. During the occlusion, all dogs had significant ischemia of the occlusion zone as measured by radiolabeled microspheres (0.08 +/- 0.08 versus 0.88 +/- 0.09 ml/g per min for the control zone), and all dogs developed systolic stretching of the ischemic zone segment. Myocardial lipid content was significantly elevated in the samples from the coronary occlusion zone (p less than 0.02). The increase in lipid signal may result from the ischemia-induced decrease in beta oxidation and resultant accumulation of fatty acyl esters (for example, fatty acids, triglycerides and acylcarnitines). In conclusion, this study shows that myocardium subjected to a brief (approximately 15 min) coronary occlusion followed by 3 h of reperfusion demonstrates a significant increase in NMR-detectable lipid content.

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.

Preliminary observations on the correlation of proliferative phenomena with in vivo 31P NMR spectroscopy after tumor chemotherapy.

The pioneering studies of Gene Cronkite, and his associates, on the methodology, theory, and reality of mammalian cell kinetics are well known. During the initial years of study of animal and human hematopoiesis, there was a tacit assumption that, in addition to dissecting the physiology of proliferating systems, we would eventually use cell kinetic techniques for the management of various hematologic and malignant conditions. This has been very slow in coming to fruition which has been disappointing to many scientists and clinicians. The reasons for it are quite clear, however, and

Detection of antineoplastic agent induced cardiotoxicity by 31P NMR of perfused rat hearts.

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.

31P-magnetic resonance spectroscopy studies of cardiac transplant patients at rest.

Studies in animal models and patients have suggested that 31P-magnetic resonance spectroscopy (MRS) may be useful in diagnosing transplant rejection, but such studies often are confounded by the late inclusion of patients after transplantation. The present study examined the utility of 31P-MRS in the diagnosis of acute allograft rejection during the first posttransplant month. Thirteen recent heart transplant recipients underwent 57 resting 31P-MRS studies within 24 hr of a biopsy. Subjects lay supine with a 10-cm surface coil placed over the heart. A 1-dimensionsal chemical shift imaging protocol was used to collect spectral information. Spectra from the heart were weighted for distance from the coil and summed before analysis. ANOVA and Duncans multiple range test were used to analyze the data comparing phosphocreatine (PCr)/ATP ratios with biopsy scores. Transplant patients had significantly lower myocardial PCr/ATP ratios when compared with a normal control group (1.27 +/- 0.27 versus 1.61 +/- 0.22, p < 0.001). However, when the patient group was classified by biopsy score, the expected order of score, 0 > 1 > 2 > 3, was not obtained. Rather, the order was 2 > 0 > 1 > 3. Although the difference between scores 2 and 3 was significant (1.46 versus 1.14, alpha = 0.05 level), the lower three groups were statistically indistinguishable. In addition, the PCr/ ATP ratios were not predictive of future biopsies. Although significantly lower than normal control subjects, resting myocardial PCr/ATP ratios of transplant subjects are not useful in assessing thelevel of rejection. It is suggested that the measurement may be more predictive in mildly exercised myocardium.