Effect of Acinetobacter Glutaminase-Asparaginase Treatment on Free Amino Acids in Mouse Tissues1

[CANCER RESEARCH 35. 1320 1325. May 1975] Effect of Acinetobacter Glutaminase-Asparaginase Treatment on Free Amino Acids in Mouse Tissues1

John S. Holcenberg, Esther Tang, and William C. Dolowy

Departments of Medicine and Pharmacology. University of Washington School of Medicine. Seattle. Washington 9X195 [J. S. H.. E. T.}. and Chicago Medical School and HiÃ±es VÃªlerons Administration Hospital. Chicago. Illinois 60612 [W. C. D.]

SUMMARY LDH virus-infected mice (26) and are confined largely to the vascular space. As expected from these considerations, Acinetobacier glutaminase-asparaginase (AGA) and treatment of mice and humans with EC-2 produces pro Escherichia coli asparaginase were compared for their longed depletion of plasma asparagine but only transient effects on plasma and tissue levels of amino acids, am depletion of plasma glutamine (21, 25). AGA treatment in monia, and glutamyl transferase activity in the mouse. Free mice produces prolonged depletion of both plasma aspara asparagine was depleted similarly in plasma and tissues by gine and glutamine (26). both enzymes. AGA treatment produced partial depletion Several studies have reported changes in free amino acids of glutamine concentrations in muscle, spleen, small intes in tissues during treatment with asparaginase enzymes, but tine, and liver. Brain and kidney glutamine concentrations none of these enzymes has had appreciable glutaminase actually rose with treatment. Despite over 100-fold increase activity under physiological conditions (2, 7, 20, 30). The in plasma glutamate, only the kidney showed a substantial amino acid levels in tissues should depend on the delivery of increase in free glutamate levels during AGA treatment. substrates and enzymes by the vascular system, together Glutamine biosynthesis measured by glutamyl transferase with the rate of synthesis and utilization of the amino acid in activity showed an appreciable increase only in the kidney. the tissue. Ammonia levels in tissues and plasma rose 1.3- to 4.3-fold. This paper compares the effect of AGA and EC-2 In general, E. coli asparaginase treatment had much less treatment on the tissue concentration of several free amino effect on these measurements than did AGA. The changes in acids, ammonia, and glutamyl transferase activity. these levels are discussed in relation to sites of possible toxicity and antitumor effects. MATERIALS AND METHODS

INTRODUCTION Materials. SSA, lithium hydroxide, lithium citrate, and ninhydrin were amino acid grade from Pierce Chemical Co., Rockford, 111.Nessler's reagent (64091) was from Harleco, Asparaginase and glutaminase enzymes have produced prolonged remissions of certain experimental tumors (40). Philadelphia, Pa. All other chemicals were reagent grade. During treatment, plasma asparagine and glutamine are AGA was isolated and purified as described previously depleted. The degree of amino acid depletion depends on the (27), except that potassium phosphate buffers were used in kinetic properties of the enzyme, its biological half-life in the final DE-Sephadex and lyophilization steps. This change the animal, and the rate of input of the amino acid into the provided much greater stability during shell freezing. In circulation. EC-23 catalyzes the hydrolysis of glutamine at addition, the sediment after sonic extraction was extracted only 3% the rate of hydrolysis of asparagine. In addition, the with 30% saturation ammonium sulfate to increase the Km for glutamine is over 100-fold that for asparagine (40). overall yield from each batch of bacteria. All AGA On the other hand, AGA has approximately equal hydro- preparations used had a specific activity greater than 120 lytic activity and an equally low Km with both amino acids lU/mg protein. Such preparations elute as a single peak on (27). The 2 enzymes have comparable biological half-lives in Sephadex G-200 chromatography and show a single band on sodium dodecyl sulfate gel electrophoresis and disc gel 1This work was supported by USPHS Research Grant C'A 11881 from electrofocusing (15). The enzyme was stored as a lyophilized the National Cancer Institute. A preliminary report of some of this work powder at 4Â°. was presented at the 65th Annual Meeting of the American Association for EC-2 was a gift from Dr. M. Zimmerman. Merck Sharp Cancer Research, at Houston. Texas. March 1974 (4). '' Research Career Development Awardee CA 25976 from National and Dohme, Inc., Westpoint, Pa., received as a lyophilized Cancer Institute. To whom reprint requests should be addressed, at powder (lot ABI-2) that had 200 lU/mg protein. This Department of Medicine RG-20. University of Washington. Seattle, Wash. preparation was homogeneous on disc gel electrophoresis 98195. (14). 3The abbreviations used are: EC-2. Escherichia coli asparaginase: Mice. CD-I female mice were supplied by Charles River AGA, Acinetobacter glutaminase-asparaginase. American Type Culture Collection No. 27197; LDH virus, lÃ¡clatedehydrogenase-elevating virus: Breeding Laboratories, Wilmington, Mass. They were fed SSA. sulfosalicylic acid. Purina mouse chow and water ad libitum. Mice were stored Received September 13, 1974; accepted February 11, 1975. 5 to 6 in a filter-top cage. Cages, water bottles, filters, and

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1975 American Association for Cancer Research. AG A Treatment Sanicel bedding (Paxton Processing Company, Paxton, 111.) to 1 ml were pipeted into 50-ml serum bottles fitted with a were steam sterilized before use. rubber stopper and a 0.3- x 6-cm ground glass rod wetted Mice were infected with LDH virus 3 or more days before with 0.5 M H2SO4. Distillation was started by addition of I treatment. Virus was initially obtained from Dr. V. Riley. ml saturated solution of K2CO3. Bottles were rotated for 20 Pacific Northwest Research Foundation, Seattle, Wash., min. The ammonium sulfate on the glass rod was rinsed into and was later propagated in mice. The dose was 0.1 ml of a a test tube with 1 ml of water and assayed with a 1:10 dilution of pooled mouse plasma in Fisher's medium concentrated phenol-hypochlorite reagent (3). Solution 1 and contained about 10" 50% infectious dose units (26). contained 6.2 g phenol and 25 mg sodium nitroprusside per Sample Preparation. Three to 4 hr after the last enzyme 100 ml. Solution 2 contained lOgNaOH and 2.15 ml bleach treatment, blood was obtained by heart puncture from mice per 100 ml. One ml Solution I and 0.2 ml Solution 2 were anesthetized with ether. The blood was treated with sodium added in rapid sequence to the samples. Tubes were heated heparin. rapidly chilled, and centrifuged. Plasma for en at 37Â°for 15 min and cooled, and the absorbance was read zyme assay was tested immediately or stored at - 100Â°. at 640 nm. Standards were NH4C1 with and without SSA. Storage for several weeks did not decrease the enzyme SSA decreased the slope of standard ammonia curves by activity. Samples for amino acid analysis were deprotein- 23%. Ammonia added to tissue samples or glutamine ized within 1 hr with 10% SSA. The precipitate was washed solutions was completely recovered, and glutamine added to 2 times with 5% SSA. The combined supernatants were tissue samples was not hydrolyzed. titrated to pH 2.6 to 2.7 with LiOH and diluted with lithium Asparaginase and glutaminase activity was determined citrate buffer, pH 2.76. by ammonia formation by direct nesslerization, as previ Tissue samples were obtained from mice anesthetized ously described (27). Glutamyl transferase activity was with ether. They were rapidly trimmed of fat, blotted dry, determined by hydroxamate formation, as previously de and weighed. The sample of small intestine was obtained scribed (33). from the 1st 8 cm of duodenum and jejunum after the lumen Treatments. All enzyme preparations were injected i.p. had been rinsed with cold 0.9% NaCl solution. Whole EC-2 and AGA were dissolved in water and administered samples or aliquots less than 0.5 g were immediately daily at doses of 250 to 350 IU/kg of body weight. Previous plunged in 2 ml 10% SSA and minced. The mince was studies showed that these maximally tolerated doses of homogenized in a Potter-Elvehjem tissue grinder with a AGA (27) maintained asparaginase activity above 1.0 Teflon plunger. The homogenate was transferred with 1 ml ID/ml of plasma. 10% SSA and centrifuged at 27,000 x g for 15 min. The pellet was rinsed with 1 ml 5% SSA and recentrifuged. The combined supernatants were titrated like the plasma sam RESULTS ples and diluted to approximately 5 ml. Exceptions to this method were the spleen, which was homogenized and Weight. The effects of enzyme treatment on animal and washed in one-half of the standard volume, and muscles, spleen weight are shown in Chart 1. AGA treatment caused which were homogenized in a Sorval Omnimix microattach- a progressive loss of up to 35% of the initial body weight by ment at full speed for 2 min. Samples were stored at - 100Â°. the 11th day of treatment. This weight loss was accompa Tissues for glutamyl transferase activity were homoge nied by marked loss of s.c. tissue and fat and moderately nized in Medium A (17) and centrifuged at 100,000 x g for ruffled hair. In contrast, animals treated with equal doses of 60 min or at 48,000 x g for 90 min (33). EC-2 based on asparaginase activity had less than 15% Amino Acid Determinations. Aliquots of plasma and weight loss, which tended to stabilize by 7 days of treatment. tissue were analyzed on a JEOLCO 5AH amino acid Spleen weight of animals treated with AGA decreased analyzer with a dual channel integrator Model DK. Neutral markedly and remained less than one-half normal on all 4 and acidic amino acids were analyzed through the glutamine days sampled. The loss of spleen weight occurred prior to peak with lithium citrate buffer, pH 2.78 to 2.84, as significant loss of body weight. AGA treatment did not formulated by Spackman (34), and JEOL resin AR-50 at significantly decrease brain or kidney weight. Control liver 39Â°.The pH of each batch of buffer was adjusted to obtain weight was 1.23 Â±0.05g (n = 9) and decreased only after 11 maximal separation of asparagine, glutamate, and gluta days of treatment to 0.77 Â±0.14 g (n = 5) (p < 0.01). mine. 7-Aminobutyric acid was analyzed on the basic Treatment with EC-2 had little effect on the weight of any of column with sodium citrate buffer, pH 4.26, and Beckman these tissues. PA35 resin at 51Â°. Glutaminase-Asparaginase Activity. Blood samples were Tissue extracts contain reduced glutathione, which chro- obtained 3 to 4 hr after the last i.p. injection. The average matographs with aspartate with our resin and buffer system. asparaginase activity in the plasma after 3, 7, and 11days of Treatment of the tissue samples with performic acid (11) to AGA treatment ranged from 2.1 to 2.4 lU/ml. Samples oxidize the glutathione markedly reduced the "aspartate" from EC-2 treatment were 5.0 and 1.8 lU/ml at 3 and 11 peak with liver, small intestine, and muscle. Kidney showed days. Enzyme activity with all treatments was high enough variable reduction. Brain and spleen showed little effect and to deplete circulating levels of asparagine from 64 Â±1Â¿/Mto therefore were the only tissues that could be evaluated for below the level of detection of the amino acid analyzer (3 to aspartate. 5 MM). Other Assays. Ammonia was determined on the sulfosali- Both enzymes should have a comparable effect on free cylic extracts used for amino acid analysis. Aliquots of 0.5 asparagine concentration in plasma and tissue. Because of

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1975 American Association for Cancer Research. ./. 5. Holcenberg et al. its greater glutaminase activity, AGA might be expected to with AGA treatment and only 2-fold with EC-2. This have a greater effect on plasma and tissue free glutamine treatment did not affect free aspartate in brain but de concentration. creased spleen levels to about 30% of control. EC-2 Asparagine. Table 1 shows the plasma and tissue free treatment did not change the levels in either tissue. asparagine levels after 11 days of AGA treatment. The Glutamine. Plasma glutamine fell from 0.55 Â±0.07mvito levels were very similar after 3 and 7 days of treatment. The undetectable levels after 3, 7, and 11 days of treatment. degree of depletion varied with different tissues. Brain free Chart 2a demonstrates several different patterns of tissue asparagine decreased only slightly. In contrast, kidney free response to depletion of circulating glutamine. Unexpect asparagine was undetectable in all samples during treat edly, the brain and kidney samples showed a progressive ment. Levels in the other tissues decreased significantly to increase in glutamine concentration to 4- and 2.5-fold by 11 less than 25% of control. EC-2 treatment produced identical days of treatment. These increases were statistically signifi effects on levels in plasma, brain, liver, kidney, and spleen. cant (p < 0.05) at the 3 sampled times. In contrast, the Aspartate. Blood aspartate rose approximately 10-fold glutamine concentration in spleen, liver, and skeletal muscle decreased to less than 25% of control by 3 days. After 11 days of treatment, the spleen level was still less than 10% 100 (0.07 Â±0.05 ^mole/g wet weight, n = 3), but the liver and skeletal muscle had returned to 60 and 90% of their respective controls. The free glutamine concentration in small intestine was only 0.29 Â±0.07/Â¿mole/g,wet weight (n 80 = 4) in control animals and fell significantly only after 7 days treatment to 0.12 Â±0.02Â¿tmole/g,wet weight (n = 5). In contrast, EC-2 caused only transient, partial depletion ÃŒ60 of plasma glutamine (0.018 HIMafter 3 days and 0.33 mM after 11 days of daily treatment). In comparison to the i nontreated controls, free glutamine in brain increased slightly to 5.78 ^moles/g at 11 days, while kidney levels decreased to 0.77 Â¿Â¿mole/gat11 days. Other tissue levels were unchanged. Glutamate. Plasma glutamate increased from 0.26 Â±

20 0.006 mvi in normal mice to over 3.5 mvi after 3, 7, and 11 369 days of AGA treatment. Simultaneously, free glutamate Doys of treolment rose to a maximum of only 3-fold in kidney (p < 0.01) and Chart I. Weight of CD-I female mice and spleen during treatment. 1.1-fold in brain (p < 0.05). Brain levels of 7-aminobutyric Body weight of each animal was expressed as percentage of its initial acid did not change during treatment. Free glutamate in weight throughout treatment. Circles, average percentage of the initial spleen actually decreased about 50% (Chart 2b). weight Â±S. E. of mean: O. animals treated with EC-2; â€¢¿.animalstreated With EC-2 plasma glutamate was 0.66 mM after 3 days with AGA. both at approximately 270 IU/kg/day i.p.: â€¢¿spleenweights during AGA treatment, expressed as percentage of the weight of untreated and 0.31 mM after 11 days of daily treatment. Liver controls. Initial weights of EC-2- and AGA-treated mice were 33.7 Â±0.7(n glutamate levels significantly increased 2-fold at 3 days, and = 4) and 27.6 Â±0.8(n = 15)g, respectively. Initial spleen weight was 0.138 brain levels increased 1.3-fold at 11 days. Free glutamate Â±0.007 (n = 9) g. levels in other tissue were not significantly changed.

Table 1 Asparagine and ammonia concentration during treatment of CD-I female mice Values expressed in j/moles/g, wet weight, represent the mean Â±S.E. of number of tissues shown in parentheses. Mice were treated for 11 days with AGA as described in the text.

Asparagine Ammonia

TissuePlasma"BrainLiverKidnevSpleenMuscleSmall

(4)"0.12Â±0.013 Â±0.001C(5)0.11,0.090.03 Â±0.03(8)2.30 Â±0.02(3)0.10 Â±0.22(6)1.52 (7)3.47Â±0.21" Â±0.01(3)0.10.0.13'0.38(4)<0.01Â±0.01' Â±0.16(5)3.56 Â±0.29'(4)8.18Â± (3)0.07 (5)2.25Â±0.14 (4)9.10Â±1.20- Â±0.06(3)0.09 (3)0.03Â±0.02r Â±0.20(5)1.74 (3)2.15Â±0.13(4)3.451.5Â» Â±0.01(5)0.33 (4)0.08Â±0.01' Â±0.16(7)2.72 intestineControl0.064Â±0.06(3)Treated<0.005 Â±0.02r(3)Control0.13.0.101.70Â±0.52(5)Treated0.58Â±0.21(4) " Plasma concentrations are in ^moles/ml plasma. 6 Numbers in parentheses, sum of animals used in the plasma pools assayed. ' Significance of difference of means of treated and control animals, p < 0.01. by Student / test. " Significance of difference of means of treated and control animals, p < 0.05, none for p > 0.05. by Student ( test. '' Only 2 samples tested.

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Glutamate

Chart 2. Tissue levels of free amino acids in CD-I female mice * during AGA treatment: a. glutamine: b. glutamate. Free amino ,t> acids Â»eredetermined by an amino acid analyzer on SSA extracts 5 of tissues during en/yme treatment. 6

Doys

Ammonia. AGA treatment raises levels of ammonia in Table 2 plasma and all tissues, as illustrated in Table 1. Maximal Effect of AGA treatment on glutam\l transferase activity of CD-I female elevation occurred between 7 and 11 days with various mice tissues and represented a 1.3- to 4-fold increase in tissues High-speed supernatant fractions were assayed for glutamyl hydroxa- and a 4.3-fold increase in plasma. EC-2 treatment produced mate formation. Activity is expressed in nmoles/min/mg protein. The 5-day treatment experiment represents the mean Â±S.E. of values from 3 a slightly higher elevation of plasma ammonia (0.73 mg/ml) animals for each tissue. but a smaller elevation in tissue ammonia of only 1- to 1.5-fold. TissueBrainLiverKidnevSpleenSmall days ofAGA482 Ammonia levels in plasma are similar to those reported in Â±27343 Â±9434 other animals (37, 39). On the other hand, ammonia levels Â±2075 Â±15Â°203 in control mouse brains are appreciably higher than those Â±10IS Â±9"26 reported previously (22, 37). Control studies indicate that Â±10.3 Â±2"0.9 these differences are not due to glutamine hydrolysis or intestineMuscleControl432 Â±0.114 Â±0.518 effect of tissue extracts on ammonia color formation. Â±25 Â±1 Serine and Threonine. Asparaginase and glutaminase " Significant differences, p < 0.05, by Student / test. have no direct effect on these amino acids. Nevertheless, " Significant differences./) < 0.01, by Student itesi. they showed a progressive increase during AGA treatment. Free threonine concentration in control tissues and plasma other tissue extracts. Other experiments showed no signifi was 0.17 to 0.55 ^mole/g wet weight. By 7 and 11 days of cant differences in tissue activity in mice with and without treatment, all tissues showed a 3- to 5.5-fold increase LDH virus infections. associated with a 2-fold increase in plasma. With EC-2 treatment, levels in plasma increased 1.5-fold and the levels in tissues increased less than 2-fold. DISCUSSION Free serine concentration in controls ranged from 0.17 in plasma to 0.99 Â¿Â¿mole/gwetweight of liver. Plasma level Treatment of experimental animals and man with as increased 1.3-fold and, in tissues, from 1.4- to 4.5-fold with paraginase enzymes has produced prolonged depletion of AGA treatment. plasma asparagine, transient depletion of plasma glutamine, Glutamine Biosynthesis. The rate of asparagine biosyn elevation of ammonia, and transient elevation of other thesis has been shown to increase following asparaginase amino acids (21, 25). AGA produces comparable depletion treatment (8, 23, 24). Experiments were designed to deter of plasma asparagine and similar elevation of ammonia. In mine whether the rate of glutamine biosynthesis is also contrast, this enzyme causes much greater and more increased during AGA treatment. Glutamine biosynthesis prolonged depletion of glutamine and a marked elevation of was measured by the glutamyl transferase reaction in glutamate in plasma (26). high-speed supernatant fractions. Table 2 shows the control In normal mice, the plasma concentration of glutamine is values and effect of AGA treatment on this activity. After 5 20 times the concentration of asparagine. In addition, days of treatment, kidney extracts showed a 2.7-fold glutamine biosynthetic activity is present in all tissues tested increase, while spleen and liver extracts showed a smaller at rates over 100 times that for asparagine. AGA, which effect. Small intestine had very low control activity that catalyzes the hydrolysis of both circulating glutamine and appeared slightly greater after AGA treatment. Separate asparagine, is bound to create a much greater load of experiments with 3 days of AGA treatment showed smaller ammonia and dicarboxylic acid products than is EC-2. As increases for activity in kidney extracts and no change with expected, this report shows that EC-2 treatment produced

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1975 American Association for Cancer Research. J. S. Holcenberg et al. much smaller effects on tissue levels of ammonia and the 8, 23, 24). On the other hand, only kidney shows an free glutamine and glutamate than did AGA. appreciable increase in glutamyl transferase activity during During AGA treatment, the tissues must synthesize AGA treatment. Perhaps the kidney enzyme is responsive to asparagine and glutamine in the presence of high-circulating increased ammonia and glutamate loads. Other kinetic ammonia, glutamate, and aspartate. The various mouse differences between kidney and other tissue glutamyl trans tissues studied appear to utilize different mechanisms for ferase enzymes have been reported (16). In the rat, gluta handling these marked dislocations. mine feeding did not change liver glutamyl transferase Excess ammonia can be eliminated by 3 pathways: (a) activity. Conflicting reports have appeared on the effect of combination with Â«-ketoglutarate to form glutamate, cata tumor growth (18, 41). Nevertheless, our data and the lyzed by glutamic dehydrogenase; (Â¿>)incorporation into previous reports suggest that mouse tissues contain suffi glutamine, catalyzed by glutamine synthesis; and (c) synthe cient transferase activity to maintain detectable free gluta sis of urea through the formation of carbamyl phosphate, mine levels during AGA treatment. primarily in the liver (32). The degree of amino acid depletion required for tumor Previous reports have shown that, in acute experiments, and normal cell death is unknown. After asparaginase large amounts of injected glutamate are taken up by brain treatment, Broome (2) showed that free asparagine in liver, tissue. This glutamate is rapidly converted to glutamine, so kidney, and both sensitive and resistant lymphomas fell to that brain glutamate remains nearly constant (10, 35). AGA less than 0.02 Â¿Â¿mole/gtissue.Cell death seemed to correlate treatment causes prolonged marked elevation of circulating with an inability of the tissue to synthesize, pool, or glutamate. Even under these conditions, the free glutamate transport sufficient asparagine. Tumors sensitive to EC-2 in brain changes very little, while free glutamine increases treatment appear to have insufficient asparagine synthetase markedly. Similarly, hepatic coma, portacaval shunting, activity to survive in the absence of circulating asparagine and experimental glutamate and ammonia loads cause (2, 8, 24). elevation of brain glutamine (36, 37, 39). Thus, the brain The basis for tumor sensitivity to glutaminase treatment can use its very high glutamine biosynthetic activity to is less clear (12). Both sensitive and resistant mouse tumors decrease both ammonia and glutamine levels. have similar glutamyl transferase activity. In the presence of Free glutamine in kidney was elevated less than in brain, asparagine, these tumors show maximal in vitro incorpora while free glutamate rose markedly. Only a small part of tion of leucine into protein at glutamine concentrations this glutamate came from blood trapped in the kidney, since greater than 0.1 mvi (12, 13). When asparagine concentra the tissue level is much higher than that in plasma. Kidney tions are less than 0.04 mM, the sensitive tumors require normally extracts plasma glutamine for production of much higher glutamine concentrations for maximal pro urinary ammonia (28, 38). In AGA-treated mice, any tein synthesis. The resistant tumors show little change in circulating glutamine is rapidly converted to glutamate, so glutamine requirement (12). Both sensitive and resistant that, in order to excrete ammonia, the kidney would have to tumor cells have intracellular free glutamine levels below take up the glutamate and ammonia and synthesize gluta 0.02 Â¿Â¿mole/gwetweight after 1 hr incubation in Roswell mine. Our observation of elevated glutamyl transferase Park Memorial Institute Medium 1640 containing 0.1 mM activity in kidney is consistent with this mechanism. glutamine (J. S. Holcenberg, P. L. Lu, and E. Tang, Liver, spleen, intestine, and muscle all showed a decrease unpublished observations). Thus, the in vivo antitumor in free glutamine with little change in glutamate. None of effect of AGA may depend on the combined depletion of these tissues appreciably increased its glutamyl transferase asparagine and glutamine to similar intracellular levels. activity. This study shows that, during AGA treatment, free Muscle normally can take up large amounts of ammonia glutamine is depleted to below 0.1 Â¿imole/gwet weight only and excrete glutamine into circulation (28). This glutamine in spleen. This organ also shows the greatest weight loss. is extracted by splanchnic tissue and used for gluconeogene- Clearly, brain and kidney maintain adequate glutamine sis and urea formation (9, 29). During AGA treatment this levels during treatment. Small intestine, skeletal muscle, cycle cannot clear ammonia, because any glutamine pro and liver show transient depletions to below 0.6 /Â¿mole/g, duced is rapidly hydrolyzed. Perhaps during treatment other wet weight. Studies of possible organ toxicity should be amino acids like alanine are utilized to transport ammonia directed at these tissues. from muscle (29, 32). Endotoxin has been demonstrated in many commercial preparations of EC-2 (19). The preparation of AGA ACKNOWLEDGMENTS includes column steps that may remove endotoxin. Never theless, part of the weight loss during enzyme treatment We wish to thank Dr. J. Roberts, Sloan-Kettering Institute for Cancer may be due to this contaminant (31). Research, New York, N.Y., and Dr. D. Spackman. Pacific Northwest Research Foundation, Seattle, Wash., for many helpful suggestions and P. The amino acid changes caused by AGA treatment Lu and B. Laine for excellent technical assistance. precede any appreciable weight loss and differ considerably from those reported during starvation and protein depriva tion (1, 6). REFERENCES Asparagine synthetase activity in various tissues increases during asparaginase treatment, nutritional deprivation of 1. Adibi, S. A., Modesto. T. A.. Morse. E. L., and Amin, P. M. Amino asparagine, and growth of asparagine-requiring tumors (2, Acid Levels in Plasma, Liver and Skeletal Muscle during Protein

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Deprivation. Am. J. Physiol., 225: 408 414, 1973. Measurement of the Brain Ammonia of the Mouse: The Effect of 2. Broome. J. D., Jr. Studies on the Mechanism of Tumor Inhibition by Glutamine on the Total Measurable Ammonia. Arch. Biochem. L-Asparaginase. J. Exptl. Med.. 127: 1055-1072, 1968. Biophys..Â«/: 377-381. 1959. 3. Chancy. A. L., and Marbach, E. P. Modified Reagents for Determina 23. Patterson. M. K., and Orr, G. R. Regeneration, Tumor, Dietary and tion of Urea and Ammonia. Clin. Chem., 8: 130 132, 1962. t-Asparaginase Effects on Asparagine Biosynthesis in Rat Liver. 4. Dolowy. W. C., and Holcenberg. J. S. Glutamine Increase in Brain of Cancer Res.. 29: 1179 1183. 1969. Mice during Treatment with Antitumor Glutaminase-Asparaginase. 24. Prager. M. D., and Bachynsky, N. Asparagine Synthetase in Proc. Am. Assoc. Cancer Res., 15: 14. 1974. Asparaginase Resistant and Susceptible Mouse Lymphomas. Bio 5. Dolowy, W. C., Roberts. J., and Holcenberg. J. S. Glutaminase chem. Biophys. Res. Commun., 31: 43 47, 1968. Induced Toxicity and Anti-glutaminase Antibodies in Mice. Proc. 25. Riley, V.. Spackman. D. H.. and Fitzmaurice. M. A. Influence of the Am. Assoc. Cancer Res., 13: 54, 1972. LDH Elevating Virus in Revealing Profound Alterations in Host 6. Felig, P.. Owen, O. E., Wahren, J.. and Cahill. G. F., Jr. Amino Acid Physiology during Asparaginase Therapy. Proc. Am. Assoc. Cancer Metabolism during Prolonged Starvation. J. Clin. Invest.. 48: Res.. 12: 64, 1971. 584 593, 1969. 26. Riley. V.. Spackman, D., Fitzmaurice. M. A., Roberts, J., Holcen 7. Frankel. D. L.. Wells. H.. and Fillios. L. C. Concentrations of berg. J. S.. and Dolowy, W. C. Therapeutic Properties of a New Asparagine in Tissues of Prepubertal Rats after Enzymic or Dietary Glutaminase-Asparaginase Preparation and the Influence of LÃ¡clale Depletion of Asparagine. Biochem. J., 132: 645 648. 1973. Dehydrogenase-elevating Virus. Cancer Res., 34: 429 438, 1974. 8. Haskell. C. M., and Canellos. G. P. L-Asparaginase Resistance in 27. Roberts. J., Holcenberg. J. S.. and Dolowy, W. C. Isolation. Human Leukemia-Asparagine Synthetase. Biochem. Pharmacol.. 18: Crystallization, and Properties of Achromohacteraceae Glutaminase- 2578-2580, 1969. Asparaginase with Antitumor Activity. J. Biol. Chem.. 247: 84 90. 9. Hills, A. G.. Reid. E. L.. and Kerr, W. D. Circulatory Transport of 1972. i-Glutamine in Fasted Mammals: Cellular Sources of Urine Am 28. Rosado. A.. Flores. G.. Mora. J.. and Soberon. G. Distribution of an monia. Am. J. Physiol., 223: 1470 1476. 1967. Ammonia Load in the Normal Rat. Am. J. Physiol.. 203: 37 42. 1962. 10. Himwich. W. A.. Petersen. J. C.. and Allen. M. L. Hematoencephalic 29. Ruderman, N. B.. and Lund. P. Amino Acid Metabolism in Skeletal Exchange as a Function of Age. Neurology. 7: 705 710. 1957. Muscle. Israel J. Med. Sci.. 8: 295 302. 1972. 11. Hirs, C. H. W. Determination of Cysteine as Cysteic Acid. Methods 30. Ryan. W. L., and Dworak. J. E. Amino Acids of the 6C3HED Enzymol.. //. 59 60, 1967. Lymphosarcoma following Treatment with Asparaginase. Cancer 12. Holcenberg, J. S.. Lu, P. L.. and Dolowy, W. C. Predictive Tests for Res., JO: 1206 1209. 1970. Tumor Sensitivity to Glutaminase-asparaginase Treatment. Proc. 31. Schmid. F. A., and Roberts. J. Antineoplastic and Toxic Effects of Am. Assoc. Cancer Res.. 15: 143, 1974. Acinelobacler and Pseudomonas Glutaminase-asparaginases. Cancer 13. Holcenberg, J. S.. Roberts, J., and Dolowy, W. C. Glutaminases as Chemotherapy Rept.. 58: 121 132. 1974. Antineoplastic Agents. In: S. Prusinerand E. R. Stadtman (eds.). The 32. Scriver. C. R., and Rosenberg. L. Amino Acid Metabolism and Us Enzymes of Glutamine Metabolism, pp. 277 292. New York: Aca Disorders. In: Major Problems in Clinical Pediatrics, Vol. 10. pp. demic Press, Inc., 1973. 77 84. Philadelphia: W. B. Saunders Co., 1973. 14. Holcenberg, J. S.. Teller, D. C., and Roberts, J. Active Enzyme 33. Shrek, R., Holcenberg. J. S.. Batra, K. V., Roberts. J., and Dolowy, Sedimentation of Antitumor Asparaginase and Glutaminase Enzymes. W. C. Effect of Asparagine and Glutamine Deficiency on Normal and Arch. Biochem. Biophys.. 161: 306 312. 1974. Leukemic Cells. J. Nati. Cancer Inst., 51: 1103 1107, 1973. 15. Holcenberg. J. S., Teller. D. C.. Roberts. J.. and Dolowy, W. C. 34. Spackman, D. H. Improved Resolution in Amino Acid Analysis in Physical Properties of Acinelobacler Glutaminase-asparaginase with Cancer Therapy Studies. Federation Proc., 28: 898, 1969. Antitumor Activity. J. Biol. Chem., 247: 7750 7758, 1972. 35. Van der Berg. C. J. Glutamate and Glutamine. In: A. Lajthe (ed.). 16. Iqbal, K.. and Ottaway, J. H. Glutamine Synthetase in Muscle and Handbook of Neurochemistry. Vol. 3, Chap. 10. New York: Plenum Kidney. Biochem. J.. 119: 145 156. 1970. Press, 1970. 17. Keller. E. G.. and Zamecnik. P. C. The Effect of Guanosine 36. Walker. C. O.. and Schenker. S. Pathogenesis of Hepatic Enceph- Diphosphate and Triphosphate on Incorporation of Labeled Amino alopathy with Special Reference to the Role of Ammonia. Am. J. Clin. Acids into Proteins. J. Biol. Chem.. 221: 45 59. 1956. Nutr., 25:619 632, 1970. 18. Knox, W. E.. Kupchik. H. Z.. and Liu. L. P. Glutamine and 37. Warren, K. S. and Schenker. S. Effect of an Inhibitor of Glutamine Glutamine Synthetase in Fetal, Adult and Neoplastic Rat Tissue. Synthesis (Methionine Sulfoximine) on Ammonia Toxicity and Me Enzyme, 12: 88 98. 1971. tabolism. J. Lab. Clin. Med., 64: 442 449, 1964. 19. Loos. M.. Vadlamudi, S., Meltzer, M., Shifrin, S., Borsos, T.. and 38. Welbourne, T. C. Ammonia Production and Pathways of Glutamine Goldin. A. Detection of Endotoxin in Commercial t.-Asparaginase Metabolism in the Isolated Perfused Rat Kidney. Am. J. Physiol., 226: Preparations by Complement Fixation and Separation by Chromatog- 544-548, 1974. raphy. Cancer Res., 32: 2292-2296. 1972. 39. Williams. A. H.. Kyu. M. H.,,Fenton. J. C. B.. and Cavanagh, J. B. 20. Miller. H. K., Krakoff, I. H., Salser, J. S., and Balis. M. E. Sensitivity The Glutamate and Glutamine Content of Rat Brain after Portacaval to L-Asparaginase and Amino Acid Metabolism. J. Nati. Cancer Inst.. Anastomosis. J. Neurochem.. 19: 1073 1077. 1972. 44: 1129 1139. 1970. 40. Wriston, J. C.. and Yellin. T. O. L-Asparaginase: A Review. Advan. 21. Miller, H. K., Salser. J. S., and Balis. M. E. Amino Acid Levels Enzymol.. 39: 185 248. 1973. following L-Asparagine Aminohydrolase Therapy. Cancer Res.. 29: 41. Wu, C., Roberts. E. H.. and Bauer, J. M. Enzymes Related to 183 187. 1969. Glutamine Metabolism in Tumor-bearing Rats. Cancer Res., 25: 22. Nathan, D. G., and Warren. K. S. A Colorimetrie Method for the 677 684, 1965.

MAY 1975 1325

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1975 American Association for Cancer Research. Effect of Acinetobacter Glutaminase-Asparaginase Treatment on Free Amino Acids in Mouse Tissues

John S. Holcenberg, Esther Tang and William C. Dolowy

Cancer Res 1975;35:1320-1325.

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