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Glutamate in the Walker 256 Carcinosarcoma in Vivo1

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Glutamate in the Walker 256 Carcinosarcoma in Vivo1 [CANCER RESEARCH 50, 4839-4844, Augu§t15.1990] Short-Term Metabolic Fate of L-[13N]Glutamate in the Walker 256 Carcinosarcoma in Vivo1 Sabina Filc-DeRicco, Alan S. Gelbard,2 Arthur J. L. Cooper, Karen C. Rosenspire,3 and Edward Nieves4 Nuclear Medicine Research Laboratory, Memorial Sloan-Ketlering Cancer Center [S. F-D., A. S. G., K. C. R., E. N.J, and the Departments of Biochemistry' and Neurology, Cornell University Medical College [A. J. L. C.], New York, New York 1002 1 ABSTRACT nitrogen derived from ammonia and amino acids in the brain (8, 9) and liver (10-12). Other laboratories have used HPLC to In vivo studies with L-[13N)glutamate in the Walker 256 carcinosar- study the fate of uN-metabolites in the myocardial septum (13, coma implanted under the renal capsule of female Sprague-Dawley rats 14) and we have previously described the metabolic fate of [I3N]- demonstrate that uptake of glutamate and the rate of incorporation of ammonia and L-[a/w/We-uN]glutamine in glutaminase-sensitive the nitrogen label from this amino acid into metabolites is slower in the and -resistant murine tumors (15). Since tracer studies with "N tumor than in nontumorous kidney tissue. Glutamate dehydrogenase, glutaminase, and alanine aminotransferase activities are significantly have provided much useful information on nitrogen turnover lower within the tumor than within the adjoining kidney. However, the in these tissues in vivo, we have adopted these procedures to tumor expresses high levels of aspartate aminotransferase, attesting to study nitrogen metabolism in an intact rat tumor model in vivo. the importance of this enzyme in the metabolism of glutamate. Indeed, Accordingly, we chose to study L-['3N]glutamate metabolism in high performance liquid Chromatographie analysis showed that the prin an implanted Walker 256 carcinosarcoma. The rationale for cipal metabolic fate of label derived from L-|13N)glutamate in the tumor the choice of the 13N-amino acid and the tumor model system is incorporation into aspartate. Measurement of specific activity ratios of is given below. glutamate to aspartate shows that the transfer of nitrogen from glutamate In vitro studies of tumor mitochondria and of tumor tissue to aspartate is rapid and that equilibration of label among components of grown in culture indicate that exogenously supplied glutamine the aspartate aminotransferase reaction is attained within minutes after is a major energy source for neoplasms (16-18). The major tumor uptake. Analyses of the nontumorous portion of the implanted kidney also showed that aspartate is the major recipient of glutamate pathway for glutamine metabolism involves conversion to glu nitrogen. However, high performance liquid Chromatographie analyses of tamate as a first step. Moreover, since glutamate can be readily labeled with I3N by an immobilized enzyme procedure, we chose deproteinized tissue revealed that glutamine and ammonia are also sig nificant "Vlabt'li'd metabolites formed from L-[13N|glutamate within the to investigate the short-term tumor metabolism of this amino kidney. Proportionately lower amounts of these labeled metabolites were acid nitrogen. To facilitate this study, we chose a rat tumor found in the tumor. model system that provides for: (a) efficient delivery of labeled glutamate; and (/>) ready quantitation of metabolites entering and leaving the implant region without perturbing or occluding INTRODUCTION blood flow. Amino acids are avidly taken up by tumors for various cellular In vitro studies performed on Ehrlich ascites carcinoma cells metabolic processes. When these compounds are labeled with by Kovacevic et al. (19, 20) demonstrated that aspartate was an a positron-emitting radionuclide such as I3N (f./,9.96 min), they important metabolite formed from the oxidation of glutamine can be used for imaging neoplasms either by positron emission and that its rate of utilization is much slower than its rate of tomography or by two dimensional scanning (1-5). Assessment synthesis. This present report details the in vivo short-term metabolic fate of label derived from L-[uN]glutamate and also with external scanning devices of changes in the concentration of label in tumors after treatment with chemotherapeutic agents emphasizes the significance of label transfer from glutamate to has been used for the design of treatment regimens for several aspartate via aspartate aminotransferase in the in situ Walker patients at Memorial Sloan-Kettering Cancer Center (6). 256 carcinosarcoma. Aspartate plays a pivotal role in supplying In addition to their value as imaging agents, l3N-labeled nitrogen for purine and pyrimidine synthesis and in the transfer compounds offer unique advantages as biochemical tracers for of reducing equivalents across the mitochondrial membrane via the study of short-term nitrogen metabolism. Nitrogen-contain the malate-aspartate shuttle. ing compounds can be labeled with high specific activity and Since a portion of the kidney was left intact and not affected can be administered in tracer doses without perturbing physio by tumor tissue growth, we also report on the short-term metabolic fate of L-[13N]glutamate in this tissue. Although logical processes. Labeled metabolites can then be rapidly sep arated by HPLC5 and quantitated by an on-line flowthrough aspartate is the major nitrogen-containing metabolite formed radioactivity detector system (7). Our laboratory has used this from glutamate, the rate of glutamate metabolism is greater protocol to determine the short-term in vivo metabolic fate of and proportionately more label is present in glutamine and ammonia in this tissue than in the tumor. Reflecting the dis Received 1/4/90; revised 4/24/90. parate metabolic requirements, marked differences were found The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in in several enzyme activities affecting glutamate metabolism as accordance with 18 U.S.C. Section 1734 solely to indicate this fact. well as in the uptake and fate of L-['3N]glutamate between the ' This work was supported in part by NIH Grants CA-34603 and DK-16739 and by Department of Energy Grant DE-FG-02-86-ER-60407. Presented in part tumor and normal kidney. at the 34th Annual Meeting of the Society of Nuclear Medicine, June 2-5. 1987, Toronto, Ontario, Canada. 2To whom requests for reprints should be addressed, at Nuclear Medicine MATERIALS AND METHODS Research Laboratory, Memorial Sloan-Kettering Cancer Center. 1275 York Avenue, New York, NY 10021. Biochemicals and Reagents. o-Phthaldialdehyde, L-lactic dehydrogen- 'Present address: PET/Cyclotron Facility, University of Michigan, Ann Ar ases from various sources (with specific activities of 500-1000 units/ bor, MI 48108. mg), L-aspartate, L-alanine, pyridoxal 5'-phosphate, a-ketoglutarate, 4 Present address: Department of Neoplastic Diseases, Mount Sinai Medical Center, New York, NY 10029. triethanolamine-HCl, EDTA-Na2H2-H2O, ammonium acetate, and 9The abbreviation used is: HPLC, high performance liquid chromatography. ADP were purchased from Sigma Chemical Company (St. Louis, MO). 4839 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1990 American Association for Cancer Research. GLUTAMATE METABOLISM IN WALKER 256 CARCINOSARCOMA NADH was obtained from Calbiochem (San Diego, ÇA).Glutamate of renal implanted tumor, nontumorous kidney, and blood were sepa dehydrogenase was purchased from Boehringer Mannheim (Indianap rately analyzed for metabolites of L-['3N]glutamate by cation exchange olis, IN) and cyanogen bromide-activated Sepharose was obtained from chromatography (Partisil SCX column, 250 x 4.6 mm) using a HPLC Pharmacia (Piscataway, NJ). on-line flowthrough radioactivity detector system (7). The SCX column i.-|13N|Glutamate Preparation. ["NJAmmonia was prepared in the was eluted with 5 mM potassium phosphate-H,PO4 (pH 2.55, at a flow Memorial Sloan-Kettering Cancer Center cyclotron (Model CS-15; rate of 1 ml/min) for 8 min and subsequently eluted with 20 mM Cyclotron Corp., Berkeley, CA) by the 16O(p,«)"N reaction on water potassium phosphate-H,POj (pH 3.5, at a flow rate of 1 ml/min) for and subsequent reduction of '\ labeled nitrates and nitrites with the remainder of the elution time (10). The column was connected Devarda's alloy (21). L-["N]Glutamate was prepared by passing a directly to a radioactivity monitor/analyzer (Ramona D; IN/US Service mixture of [13N]ammonia, a-ketoglutarate, and NADH through a col Corp., Fairfield, NJ) by a 6-port valve. Data received by the detector umn on which glutamate dehydrogenase had been immobilized to a were decay corrected, plotted, analyzed, and stored by a separate solid support of CNBr-activated Sepharose (22). computer program (ISOMESS, Straubenhardt. West Germany). Me Experimental Animals and Tumor Implants. Walker 256 carcinosar- tabolite elution profiles were compared with the profiles of known coma was obtained from the National Cancer Institute (Bethesda, MD). nonradioactive standards. Female Sprague-Dawley rats (HaríanSprague-Dawley, Madison, WI) Analysis of Unlabeled Amino Acids and Urea ¡nTissues.Frozen tissue weighing 250-375 g were anesthetized with an i.m. injection of 4-6 samples were pulverized to a fine frozen powder in a cryostat. Known mg/100 g body weight of ketamine HC1. To assure the effectiveness of amounts of »-aminoadipate were added, as an internal standard, to the the anesthesia, the animals were supplemented with ether
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