Daunorubicin Metabolism by Human Hematological Components1

[CANCER RESEARCH 32, 600-605, March 1972)

David H. Huffman2 and Nicholas R. Bachur3

Biochemistry Section, Laboratory of Pharmacology, Baltimore Cancer Research Center, National Cancer Institute, USPHS Hospital, Baltimore, Maryland 21211

SUMMARY MATERIALS AND METHODS

Daunorubicin, an antitumor antibiotic that is widely used in Chemicals. All solutions were prepared in glass-distilled the treatment for acute leukemia, is converted to demineralized water, and solvenls were reagenl grade. NADPH daunorubicinol by a cytoplasmic enzyme, daunorubicin was purchased from PL Biochemicals, Madison, Wis., and Dl reducÃase, in rat tissues. We find that intact human blood was oblained from Ihe Drug Developmenl Branch of Ihe elements, as well as homogenates, can catalyze this reaction. In Cancer Chemolherapy National Service Cenler, Nalional both leukocytes and erythrocytes, enzymatic activity is Cancer Inslilule, NIH, and was purified before use (4). cytoplasmic and heat labile, requires NADPH, and does not Tissue Preparations. All tissue preparations were made in require molecular oxygen. Of formed blood components, the cold. Freshly drawn, heparinized, human venous blood was specific activities (per mg protein) rate as follows: lymphocyte used for all experiments except in the platelet preparations, in > whole WBC > bone marrow > RBC > platelet. No activity is which citrale was used for Ihe anticoagulant. detected in cell-free plasma. The characteristics of Erythrocytes. The RBC's were sedimented from whole daunorubicin reducÃase show it to be a constilutive enzyme blood by centrifugalion at 500 X g for 10 min. The unlike the classic drug-metabolizing syslem. sedimenled cells were resuspended and resedimented twice in iced 0.9% NaCl solulion before use. Whole cells were prepared fresh daily. RBC ghosls were isolated in 2 fractions, as INTRODUCTION described by Wins and Schoffeniels (32). Leukocytes. Whole WBC's were isolated according to the In the Irealmenl of acule lymphocytic and acute technique of FallÃ³n et al. (14), except that our initial granulocytic leukemia, daunorubicin (daunomycin, sedimentation was without dextran. Siliconized glass was used rubidomycin, rubomycin C), is currently one of the mosl Ihroughoul and eliminated the necessily of adding aclive agenls for inducing remissions (8, 9). This anlibiolic is streptokinase and streptodornase. After hypotonie lysis of produced by Streptomyces peucetius (17) and Streptomyces RBC contamination, the WBC's were greater than 95% viable coeruleorubidus (13) and apparenlly acls through interference (trypan blue exclusion) and essentially free of RBC (<1%) wilh nucleic acid melabolism (31). Since we have reporled and platelet contaminalion. Differential counts of ine WBC that several rat tissues metabolized Dl4 (4), and the product isolate closely approximated the inilial convenlional of this metabolism is found in human urine (2), the possibility differential count. This was in agreemenl wilh olhers who used was slrong that human tissues could metabolize this cytotoxic a similar melhod (19). For oblaining lymphocyles, anthracycline glycoside. polymorphoneulrophils were removed from Ihe above In rat tissue preparations, Dl is converted lo D2 by a preparations according to the melhod of Cooper (11). Platelet cyloplasmic enzyme, DI reducÃase,wilh NADPH as a cofactor isolation was performed by the method of Moake et al. (27), (3, 5). This reaction, as well as the struclures of Dl and D2, but withoul washing in mannilol, Tris, or EDTA. are given in Chart 1. We find thai human blood elements can Homogenales of Ihe above cell preparations were prepared metabolize Dl through this palhway, and describe our findings in 2 volumes of 0.05 M Tris-Cl (pH 7.4) wilh a conical, in this report. ground-glass homogenizer and slored al -20Â° until use. Only once-lhawed homogenales were assayed. For removal of 1A preliminary report of this work was presented at the Meeting of sedimenting material after thawing, the homogenates were the American Federation for Clinical Research, May 3, 1970, in centrifuged al 2,000 X g for 5 min al 4Â°before use. Where Atlantic City, N. J. "Present address: Clinical Pharmacology Study Unit, University of indicated, the 105,000 X g supernatant solutions were Kansas Medical Center, Kansas City, Kan. 66103. prepared by centrifuging fresh homogenates in a Beckman 3To whom reprint requests should be addressed at the Biochemistry Model L ultracentrifuge for 60 min. Section, Laboratory of Pharmacology, Baltimore Cancer Research Daunorubicin Metabolism by Tissue Preparations. In Center, National Cancer Institute, USPHS Hospital, 3100 Wyman Park whole-cell experiments, 1.5X IO8 cells (RBC, WBC) were Drive, Baltimore, Md. 21211. 'The abbreviations used are: Dl, daunorubicin; D2, daunorubicinol; incubaled in Krebs-Ringer bicarbonale lhal conlained 0.523 S-FU, 5-fluorouracil. /imole DI in a final volume of 0.5 ml. The incubations were Received September 29, 1971 ; accepted December 8, 1971. done in stoppered, pregassed (95%O2 - 5%C02) tubes in a

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NH2H

DAUNORUBICIN (DI) DAUNORUBICINOL (D2) Chart 1. DI reducÃasereaction.

Table 1 aglycones into toluene. The hydrolyzed product, D2 aglycone, D2 production by blood components from normal subjects was separated from the substrate Dl aglycone by silicic acid See text for details of incubation mixture and assay procedure. In microcolumns. Quantification of D2 aglycone in the elution the absence of NADPH, no detectable D2 production occurred with solution was by fluorescence analysis in a Baird Atomic any preparation. spectrophotofluorimeter (SF-1) (activation, 470 nm; emission, 10 -3 iimole/ 585 nm). The nmoles of D2 formed per 30 min were related to TissuepreparationRBC (mg)1.050.460.801.72.14.0D2(Xmgprotein)1 the protein concentration determined by the method of Lowry et al. (25) to obtain the specific activity of the homogenateWBC .84 Â±0.44Â°9.8 homogenateWBC Â±2.02b10.8C20.619.71.7"2.15NDCpreparations. homogenateLymphocyte homogenatePlatelet RESULTS homogenateBone homogenatePlasmaProteinmarrow When whole WBC's and RBC's were incubated with Dl in Krebs-Ringer bicarbonate, D2 was the only metabolite observed. Although the WBC's appeared to produce more " Mean Â±S.D.of 14 normal subjects. metabolite than RBC's, the evaluation of Dl metabolism by b Mean Â±S.D.of 14 normal subjects. c This WBC preparation was the source of the lymphocyte whole WBC's was complicated by cell clumping under the preparations. conditions used. Since D2 was the only metabolite observed in d Platelets pooled from 10 units of blood. the intact RBC's and WBC's, we decided to measure the e ND, no detectable metabolite production, i.e., less than 0.005 X IO"3 Mmole D2 per mg protein was observed. metabolism of Dl in cell homogenates and fractions in order to circumvent problems of permeability, drug transport, cell agglutination, etc. metabolic shaker at 37Â°for 30 min. Extraction of the reaction D2 production levels by RBC and WBC homogenates are mixture and thin-layer chromatography were carried out as given in Table 1. The mean value for WBC homogenates was 5 previously described (4). times higher than the RBC value obtained from the same 14 For homogenate and cell fraction experiments, the reaction patients. There was no correlation between level of catalytic mixture contained 0.523 Â¿/moleDl, l jumÃ³leNADPH, 50 activity and either age or sex of the subjects. Aimoles Tris-Cl (pH 7.4), 0.4 to 0.8 mg protein for WBC, and 3 When the blood components were isolated and studied for to 4 mg protein for RBC in a final volume of 0.5 ml. The metabolic activity, we found that lymphocyte homogenates assays were performed in duplicate under room air in a showed the highest specific activity (Table 1). The lymphocyte metabolic shaker at 37Â°for 30 min. The reaction was started homogenates were assayed simultaneously with whole-leuko by the addition of homogenate and terminated with 2.0 ml 0.3 cyte homogenates isolated from the same patient. In each case, N HC1 in 50% ethanol. The conditions for the reaction were the specific activity of the lymphocyte homogenate was twice selected from experimental data presented in "Results." the specific activity of the leukocyte homogenate, indicating a Assay for D2 production has been described in detail (6). higher specific activity in the lymphocyte preparations than in Briefly, this involved acid hydrolysis of the Dl and D2 other white cell types. None of the preparations showed glycosidic bonds followed by extraction of the nonpolar detectable activity in the absence of NADPH.

MARCH 1972 601

Table 2 p-nitrophenylphosphate according to the method of Ahmed Subcellular localization of Dl reducÃasein blood cells and Judah (1). Phosphatase activity (Table 3) was present in Results are the average of duplicate determinations on the same both fractions, and the less vigorously treated "SI fraction" homogenate. In each case the 105,000 Xg supernatant was prepared retained the labile K+-stimulated phosphatase activity. Neither from and analyzed simultaneously with the whole-homogenate of these membrane fractions metabolized Dl. solution. The RBC ghosts were prepared according to the method of Wins and Schoffeniels (32), whereas the WBC particulate fraction was As demonstrated in Tables 1 and 2, NADPH was a required the unwashed sedimenting fraction from the ultracentrifuge cofactor for activity in all the homogenates (2 mM NADH preparation. See text for details of the incubation mixture and assay showed about 13% of the cofactor activity that NADPH procedure. In the absence of NADPH, no detectable D2 production showed with leukocyte homogenates). NAD and NADP, occurred with any preparation. however, failed to support metabolite production by the (X 10 "3 ornÃ³le/ whole-leukocyte homogenate. When NADPH was added in TissuepreparationWBCWhole (mg)1.050.6241.54.84.31.04D2mg protein)10.7521.51.42.31.6NDÂ°increasing concentrations to the reaction mixture, it was found that 0.1 mM was saturating (Chart 2). No substrate inhibition was found at 3 mM, so that a final concentration of 2 mM homogenate105,000 supernatantParticulateX g NADPH was used in all analyses. fractionRBCWhole When Dl was added in increasing concentrations to normal leukocyte homogenate, maximum metabolite production occurred at 3.4 X 10~4 M Dl (Chart 3). At higher substrate homogenate105,000 supernatantRBCX Â£ concentrations studied, there was no inhibition of D2 ghostsProtein production. For this reason and to obtain easily measurable levels of product, the substrate level at 1.046 mM was selected. 0 ND, no detectable metabolite production, i.e., less than 0.005 X IO"3 ÃimoleD2 per mg protein was observed. D2 production was linear for protein concentration and time up to 30 min with WBC whole homogenates (Chart 4) under the selected conditions. RBC homogenates demonstrated Table 3 similar characteristics with regard to NADPH and Dl Enzymatic activities of isolated RBC ghosts" saturation, as well as linearity to protein concentration and time. (jumÃ³les When either WBC or RBC homogenates were heated at 56Â° split/mg protein)0Mg** for 2 min, 85% of the catalytic activity was lost. When they (X 10 "3 Mmole/ were heated for 15 min, the activity was completely PreparationSI mg protein)bNDd *0.0725Mg" plus K destroyed. Dialysis against 1000 volumes 0.05 M Tris-Cl (pH 7.44) for 4 hr did not reduce catalytic activity as long as fraction 0.0918 NADPH was supplied as cofactor. Similar to previous S2 fractionD2 NDPhosphate 0.035 0.030 observations on rat tissue preparations, the production of D2 " RBC ghosts prepared according to the technique of Wins and by WBC and RBC homogenates proceeded unaffected under Schoffeniels (32). nitrogen. With standard assay conditions, a RBC homogenate b Dl metabolism evaluated according to technique described in (1 mg protein) produced 2.0 X 10~3 /Â¿moleofD2 under air or "Materials and Methods." c Phosphatase activity: reaction mixture contains 5 mM MgCl2, 100 mM Tris-Cl, 5 mM p-nitrophenyl phosphate, 2 to 3 mg protein and, where indicated, 20 mM KG. Incubation was for 30 min at 37Â°. Analysis of enzymatic activity was according to the technique of Ahmed and Judah (1). 10.0 d ND, no detectable metabolite production, i.e., less than 0.005 X IO"3 Mmole D2 per mg protein. o er CL O The bulk of catalytic activity was found in the 105,000 X g s supernatant of both RBC and WBC homogenates (Table 2). It was likely that the small amount of catalytic activity found in 5.0 (M the WBC residue represented contamination by the O supernatant fraction. Similarly, the catalytic activity was also found in the 105,OOOXÂ£ supernatant of the RBC o homogenate, with no activity in the RBC ghost preparation. Z The activity of all the fractions was totally dependent on NADPH. The decrease in activity of the high-speed RBC supernatant compared with the whole RBC homogenate is 1.0 2.0 3.0 [NADPH] x IO'3 M unexplained. To ensure that the RBC ghost preparations that showed no Chart 2. The effect of NADPH concentration on D2 production by detectable reducÃase activity did show other enzymatic normal WBC homogenate. See "Materials and Methods" for details. competence, they were tested for their ability to hydrolyze D2 X 10"3 = nmoles D2.

602 CANCER RESEARCH VOL. 32

in the 105,000 Xg supernatant of both WBC and RBC homogenates, as well as in other tissues (5), and is most likely cytoplasmic in location. This subcellular localization is further supported by the absence of detectable activity in RBC ghosts, despite the presence of the marker enzyme, p-nitrophenylphosphatase, in these preparations. Therefore, the production of D2 by blood cells is enzymatic, indicating the presence of DI reducÃasein normal human blood and bone marrow. In these hematological tissues, D2 is the only metabolite observed. Earlier studies from our laboratory indicated that rat tissue slices (4) or homogenates (5), especially from brain, liver, and kidney, not only produced D2 but also produced aglycone metabolites. The blood cells under the conditions tested do not hydrolyze the glycosidic linkage. Di Marco et al. (12) also observed aglycone production by rat kidney and liver preparations. The erythrocyte can metabolize Dl; this is especially l.O 20 -3 unusual since only a few other examples of drug metabolism DAUNORUBICIN (DI) X IO M by erythrocytes have been reported (15, 18). In addition, Chart 3. The effect of Dl concentration on D2 production by although metabolism by the platelets of naturally occurring normal WBC homogenate. See "Materials and Methods" for details. pharmacological substances such as dopamine is known (29), timÃ³leD2 X IO'3 = nmoles D2. the demonstration of the metabolism of a foreign molecule such as Dl by platelets is unique. For homogenates of the various blood components, the rank order of specific activity (per mg protein) is lymphocyte > whole WBC > bone marrow > RBC > platelet with no detectable activity in plasma. The patterns of metabolism and the nature of the various enzymes in leukocytes have been the subject of several recent reviews (10, 14, 21). Several dehydrogenases have been characterized in circulating leukocytes. Decreasing enzymatic activity of 3 enzymes (isocitrate dehydrogenase, glucose 6-phosphate dehydrogenase, and 6-phosphate-gluconate dehydrogenase) are as follows: eosinophil > neutrophil > myelocyte > myelo- blast> leukemic lymphocyte and normal lymphocyte (7, 16). In addition, other investigators have reported that overall glycolytic enzyme activity in mature lymphocytes of chronic lymphocytic leukemia is lower than that of the normal 0 1.0 2.0 leukocyte population (16, 26). In this regard, it is significant MG PROTEIN that lymphocytes show a higher specific activity of Chart 4. Effect of time and protein concentration on D2 production daunorubicin reducÃase than other blood cells. This is similar in normal WBChomogenate. /amolÃ©D2X IO"3 = nmoles D2. to the demonstration of higher 1-0-D-arabinofuranosyl cytosine (cytosine arabinoside) phosphorylation in normal nitrogen, whereas a WBC homogenate (1 mg protein) produced lymphocytes (23). 13.6 X 1(T3 /Limoleof D2 under air and 16.2 X IO'3 /Limole The earlier studies (7, 16, 26) base the enzymatic activity D2 under nitrogen. and lÃ¡clate production on a per cell basis. The relationship of metabolic activity to protein concentration in this study and the cytosine arabinoside study (23) in part circumvents the DISCUSSION difficulty in comparing enzymatic activity in diverse cell populations by expressing specific activity per mg protein We demonstrated that Dl is metabolized by cellular concentration. This is particularly importanl when there is components of normal human blood and bone marrow. The considerable variation in nuclear:cytoplasmic ratio, which metabolic activity that exists in whole blood cells as well as in exist in leukocytes or when cell size varies. homogenates is an unusual example of drug metabolism, since A number of anticancer drugs have been evaluated for their Dl is not a structural analog of a known, normally occurring effect on the metabolism of both normal and leukemic metabolite. leukocytes, and attempts have been made to correlate drug The catalytic activity is heat labile and nondialyzable, and effects to clinical response. Well studied were amethopterin, requires NADPH but not O2 for activity. Moreover, it is found cytosine arabinoside, and 5-FU. Although amethopterin and

MARCH 1972 603

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1972 American Association for Cancer Research. David H. Huffman and Nicholas R. Bachur cytosine arabinoside affected several leukocyte enzymes, these Daunomycine. Paris, HÃ´pital Saint Louis, 11 Mars 1967. Pathol. effects showed poor correlation to clinical response (28). Biol., 75. 921-924, 1967. These variations in clinical response might be explained by 10. Cline, M. J. Metabolism of the Circulating Leukocyte. Physiol. Rev., 45: 674-720, 1965. individual differences in drug metabolism. For example, 11. Cooper, H. L. Ribonucleic Acid Metabolism in Lymphocytes cytosine arabinoside is phosphorylated to an active form by Stimulated by Phytohemagglutinin. II. Rapidly Synthesized deoxycytidine kinase. Some correlation between the kinase Ribonucleic Acid and the Production of Ribosomal Ribonucleic levels and the susceptibility of the cell to cytosine arabinoside Acid. J. Biol. Chem., 243: 33-43, 1968. has been reported (23). Also, deoxycytidine deaminase has 12. Di Marco, A., fioretti, G., and Rusconi, A. Metabolic been described, and preliminary investigations suggest that the Transformation of Daunomycin by Tissue Extracts. Farmaco deoxycytidine:deaminase-kinase ratio may be very critical in (Pavia) Ed. Sci., 22: 535-542, 1967. 13. Dubost, M. P., Ganter, P., Maral, R., Ninet, L., Pinnert, S., Preud' predicting clinical response to cytosine arabinoside (30). Similarly, the antimetabolite 5-FU is converted to homme, J., and Werner, G. H. A New Antibiotic with Cytostatic 5-fluoro-2'-deoxyuridine 8'-monophosphate by tumor cells, Properties. Cancer Chemotherapy Rept., 41: 35-36, 1964. and the capacity of the tumor cells to metabolize 5-FU to this 14. FallÃ³n, H. J., Frei, E., Davidson, J. D., Trier, J. S., and Burk, D. active form appears correlated with their sensitivity to 5-FU Leukocyte Preparations from Human Blood: Evaluation of Their Morphological and Metabolic State. J. Lab. Clin. Med., 59: (24). A subline P388 murine leukemia cell, which is sensitive 779-791, 1962. to 5-FU, has shown low phosphokinase levels, however, thus 15. Fox, I. H., Wyngaarden, J. B., and Kelley, W. N. Depletion of suggesting that nucleotide formation is not the only Erythrocyte Phosphoribosyl Pyrophosphate in Man, a Newly determinant of 5-FU responsiveness (22). Observed Effect of Allopurinol. New Engl. J. Med., 283: Dl is capable of being metabolized by the cells that are the 1177-1182, 1970. target of its therapeutic effect, the leukemic myeloblasts (20). 16. Ghiotto, G., Perona, G., DeSandre, G, and Cortesi, S. Hexokinase Once formed, D2, which is the major in vivo metabolite, shows and TPNH Dependent Dehydrogenases in Leukocytes in Leukemia a longer plasma and urinary half-life in man than the parent and Other Hematological Disorders. Brit. J. Haematol., 9: 345-350, 1963. compound, Dl (20). Since D2 is effective as a cytotoxic agent 17. Grein, A., Spalla, C., Di Marco, A., and Canevazzi, G. Descrizione e in LI 210 cell tissue culture (N. R. Bachur and R. H. Adamson, Classificazione di un Attinomicete (Streptomyces peucetius sp. personal communication), its presence in the cell may be nova) Produttore di una Sostanza ad Attivitta Antitumorale: la critical to overall drug effect. Since leukemic myeloblasts Daunomicina. Giorn. Microbio!., 77: 109-118, 1963. metabolize Dl to D2, we are currently investigating the 18. Henderson, R. J., Jr., Hill, F. L., and Mills, G. C. Phosphorylation possibility that the therapeutic effect of Dl in acute leukemia of Tris(hydroxymethyl)aminomethane by Human Erythrocytes. might be related to its metabolism. Arch. Biochem. Biophys., 139: 311-319, 1970. 19. Huennekens, F. M., Yu, W. L., Calley, W. D., and Kellock, G.. Biochemistry of the leukocyte (with Special Reference to REFERENCES Dihydrofolate ReducÃase).Ser. Haematol., 10: 61, 1965. 20. Huffman, D. H., Benjamin, R. S., and Bachur, N. R. Daunorubicin 1. Ahmed, K., and Judah, J. D. Preparation of Lipoproteins Metabolism in Acute Myelogenous Leukemia. Clin. Res., 79: 493, Containing Cation-dependent ATPase. Biochim. Biophys. Acta, 93: 1971. 603-613, 1964. 21. Karnovsky, M. L. The Metabolism of Leukocyles. Seminars 2. Alberts, D. S., Bachur, N. R., and Holtzman, J. L. The Hemalol.,5: 156-165, 1968. Pharmacokinetics of Daunomycin in Man. Clin. Pharmacol. 22. Kessel, D., Hall, T. C., and Reyes, P. Metabolism of Uracil and Therap., 72:96-104, 1971. 5-Fluorouracil in P388 Murine Leukemia Cells. Mol. Pharmacol., 5: 3. Bachur, N. R. Daunorubicinol, a Major Metabolite of 481-486,1969. Daunorubicin. Isolation from Human Urine and Enzymatic 23. Kessel, D., Hall, T. C., and Rosenthal, D. Uptake and Reactions. J. Pharmacol. Exptl. Therap., 777: 573-578, 1971. Phosphorylation of Cytosine Arabinoside by Normal and Leukemic 4. Bachur, N. R., and Cradock, J. R. Daunomycin Metabolism in Rat Blood Cells in Vitro. Cancer Res., 29: 459-463, 1969. Tissue Slices. J. Pharmacol. Exptl. Therap., 775: 331-337, 1970. 24. Kessel, D., Hall, T. C., and Wodinsky, I. Nucleotide Formation as a 5. Bachur, N. R., and Gee, M. Daunorubicin Metabolism by Rat Determinant of 5-Fluorouracil Response in Mouse Leukemia. Tissue Preparations. J. Pharmacol. Exptl. Therap., 777: 567-572, Science, 154: 911-913, 1966. 1971. 25. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 6. Bachur, N. R., and Huffman, D. H. Daunorubicin Metabolism: Protein Measurement with the Folin Phenol Reagent. J. Biol. Estimation of Daunorubucin ReducÃase.Brit. J. Pharmacol., 43: Chem., 193: 265-275, 1951. 828-833, 1971. 26. Miwa, S., Tanaka, K. R., and Valentine, W. N. Erythrocyte and 7. Beck, W. S. The Control of Leukocyte Glycolysis. J. Biol. Chem., Leukocyte Glycolytic Enzyme Studies in HÃ©matologieand 232: 251-270, 1958. Nonhematologic Diseases. Acta Haematol. Japon., 25: 12-26, 8. Bernard, J., Boiron, M., Jacquillat, A., Najean, Y., Weil, M., and 1962. Thomas, M. Traitement des LeucÃ©miesAiguÃ«sLymphoblastiques 27. Moake, J. L., Ahmed, K., Bachur, N. R., and Gutfriend, D. H. de PremiÃ¨re Atteinte par une Association de Prednison- Mg2'-dependent (Na* + K*)-stimulated ATPase of Human Platelets. Vincristine-Rubidomycine. Colloque International sur la Rubiodo- Properties and Inhibition by ADP. Biochim. Biophys. Acta, 277: mycine et la Daunomycine. 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29. Solomon, H. M., Spirt, N. M., and Abrams, W. B. The 31. Theologides, A., Yarbo, J. W., and Kennedy, B. J. Daunomycin Accumulation and Metabolism of Dopamine by the Human Inhibition of DNA and RNA Synthesis. Cancer, 21: 16-21, 1967. Platelet. Clin. Pharmacol. Therap., //. 838-845, 1970. 32. Wins, P., and Schoffeniels, E. S. The Association of Some 30. Steuart, C. D., Burke, P. J., and Owens, A. H., Jr. Correlation of Oxidoreductases with the Red Cell Membrane. Biochim. Biophys. ara-C Metabolism and Clinical Response in Acute Leukemia. Proc. Acta, 185: 287-296, 1969. Am. Assoc. Cancer Res., 12: 89, 1971.

MARCH 1972 605

David H. Huffman and Nicholas R. Bachur

Cancer Res 1972;32:600-605.

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