[CANCER RESEARCH 27, 1312-1315,July 1967] Sphingolipid Metabolism in Leukemic Leukocytes1 JOHN P. KAMPINE, ROSCOE O. BRADY,- RONALD A. YANKEE, JULIAN N. KANFER, DAVID SHAPIRO, AND ANDREW E. GAL Laboratory of Neurochemislry, National Institute of Neurological Diseases and Blindness, the Medicine Branch, National Cancer Institute, Bethesda, Maryland ¿0014,and Department of Organic Chemistry, Weizmann Institute of Sciences, Rehovoth, Israel SUMMARY It has been shown that white blood cells contain sphingolipids (18), and recently we have discovered that leukocytes contain The activity of several enzymes in white blood cells, which catalyze the hydrolysis of sphingolipids and of p-nitrophenyl- very active enzymes which catalyze the hydrolysis of several sphingolipids (14). Attenuation of the activity of glucocerebro phosphate, was determined in leukocyte preparations obtained side-cleaving enzyme in various tissues has been identified as the from hematologically normal individuals and was contrasted metabolic defect in Gaucher's disease (6) and sphingomyelin- with the level of activity of these enzymes in preparations from cleaving enzyme in Niemann-Pick disease (7). The observation patients with acute and chronic leukemia and other diseases. A marked increase in the activity of glucocerebroside-cleaving en that these enzymatic deficiencies are also present in circulating leukocytes has formed the basis of a recently developed diagnos zyme was observed in leukocyte preparations from the patients tic test for sphingolipodystrophies (14). with acute and chronic myelogenous leukemia compared with the controls and patients with lymphocytic leukemia. Sphingo- In view of the presence of these enzymes in leukocyte prepara myelin-cleaving enzyme activity was elevated in leukocytes ob tions, it was considered of interest to examine the level of ac tivity of these sphingolipid hydrolases in leukocyte preparations tained from some of the patients with myelogenous leukemia. Galactocerebroside-cleaving enzyme activity and acid phos- from patients with acute and chronic myelogenous and lympho cytic leukemia. Striking differences in the level of activity of phatase were not elevated in cells obtained from any of the glucocerebroside-cleaving enzyme, and to a lesser extent, sphingo- patients. The major portion of the glucocerebroside-cleaving myelin-cleaving enzyme were observed in the preparations from activity of white blood cells was associated with the acid- phosphatase-rich subcellular granular fraction. patients with myelogenous leukemia compared with leukocytes from normal controls and from patients with various other condi tions. The nature and extent of these changes are described in the INTRODUCTION present communication. Studies of normal and loukemic white blood cells have shown that their metabolic activities vary with the different morphologic MATERIALS AND METHODS cell types (23) and can be influenced by phagocytosis (16) and by various hormones and drugs (2, 9, 17). Alterations in enzyme Leukocytes were obtained from normal volunteers and patients activities of leukemic white blood cells have been described for with acute and chronic leukemia and other diseases at the Clini enzymes involved in phosphate, carbohydrate, lipid, protein, and cal Center of the NIH. Circulating leukocyte counts ranged from nucleic acid metabolism (9). Altered alkaline phosphatase ac 1,100 to 320,000 cells per cubic millimeter. tivity of leukocytes in chronic myelogenous leukemia has been re Preparation of Leukocytes. Ten-nil samples of venous blood lated to a change in enzyme content of individual neutrophils were mixed with 2 ml of a solution containing 5% Dextran (9). More recently, studies of specific cell types have shown that (w/v), 0.7% sodium chloride (w/v), and 0.5 mg of heparin per changes in the activity of DNA polymerase and of various en ml. The cells were allowed to settle for 45 minutes at room tem zymes involved in the metabolism of glucose may be related perature, and the upper phase consisting mainly of white blood primarily to changes in the differential count (19). cells and plasma was transferred with a capillary pipette and Complex lipids represented by the sphingolipids have been re centrifuged at 600 X g for 10 minutes. The leukocyte pellet was ported to play an important role in cell structure (22). Some resuspended in 1 ml of 0.85% (w/v) sodium chloride solution and members of this class of lipids have been implicated as receptors residual red cells were lysed by the addition of 3 volumes of dis for drug action (24). Other studies have indicated that specific tilled water. After 90 seconds, 1 volume of 3.6% sodium chloride sphingolipids may be involved in cellular immune reactions (3, was added to return the cell suspension to isotonicity (12). The 13), especially in rapidly growing normal cells and cancer cells suspension was then centrifuged and the pellet washed twice with (20,21). 0.85% sodium chloride solution. Whole leukocyte suspensions in saline were used in the assays for glucoeerebroside and galacto- cerebroside-cleaving enzyme activity. The suspensions were ad 1A portion of this work was performed under Section 104 (K) justed to a final concentration of leukocytes ranging from 1-6 X of Public Law 480, 83rd Congress, Agreement No. 42515. IO7leukocytes per ml as determined with a Coulter counter. The *Address requests for reprints to Dr. Roscoe O. Brady, NIH, Bethesda, Md. 20014. activity of glucocerebroside-cleaving enzyme is proportional to Received January 4, 1967; accepted March 28, 1967. the white cell concentration in this range (14). LSI2 CANCER RESEARCH VOL. 27 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1967 American Association for Cancer Research. Sphingolipid Metabolism in Leukemic Leukocytes In order to determine the subcellular distribution of the gluco m/imoles of sphingomyelin-14C, 50 /ig of sodium cholate, 25 cerebroside-cleaving enzyme, white blood cells were homogenized /imoles of potassium acetate buffer (pH 5.0), and water in a final in 5 volumes of 0.34 Msucrose using a motor-driven homogenizer volume of 0.18 ml. The mixtures were incubated for 1 hour at with a Teflon pestle. Subcellular fractions were prepared by 37°Cand the amount of phosphorylcholine-14C cleaved was de differential centrifugation according to Cohn and Hirsh (10) ex termined in the manner indicated for the assay of the cerebroside- cept that the second centrifugation was carried out at 8700 X g. cleaving enzymes. Sphingomyelin-cleaving enzyme activity was assayed in a solu The acid phosphatase activity of various leukocyte prepara ble enzyme preparation obtained by sonicating leukocytes sus tions and subcellular fractions was determined according to Cohn pended in 5% (w/v) sodium cholate solution according to the and Hirsch (10) except that p-nitrophenylphosphate was em method described previously (14). ployed as the substrate. Enzymatic Assays. The enzymes which catalyze the hy drolysis of glucocerebroside, galactocerebroside, and sphingo- RESULTS myelin were assayed using 14C-labeled substrates. The synthesis of glucocerebroside and galactocerebroside labeled in carbon 1 of The level of activity of sphingolipid-hydrolyzing enzymes and the hexose portion of the molecule and sphingomyelin labeled in acid phosphatase in leukocytes obtained from normal donors and methyl carbon atoms has been described in earlier communica patients with various diseases is shown in Table 1. The activity tions (5, 8, 15). The enzymatic hydrolysis of the cerebrosides was of these enzymes in patients with acute and chronic myelogenous determined by incubating 50- to lOO-^ilaliquots of the leukocyte leukemia is indicated in Table 2. Of particular interest is suspensions with 125 m/mioles of labeled cerebroside, 300 pg of the higher level of activity of the glucocerebroside-cleaving en sodium cholate, 200 /¿gof Cutscum (isooctylphenoxypolyoxy- zyme in the leukemic leukocyte preparations. The mean value of ethanol, Fisher Chemical Co.), and either 15 /miólesof potassium glucocerebroside-cleaving activity in the control series was phosphate buffer (pH 6.0) for glucocerebroside-cleaving enzyme 4.6 m/iinoles of substrate hydrolyzed per mg of leukocyte protein or 15 //moles of potassium acetate buffer (pH 5.0) for galacto- per hour. Whereas, in preparations from patients with myelog cerebroside-cleaving enzyme. The final volume was brought to enous leukemia, both acute and chronic, the mean value for the 0.15 ml with water. After incubation for 1 hour at 37°Cin air, activity of glucocerebroside-cleaving enzyme was 12.8 m/umoles. the amount of glucose-l-I4C or galactose-l-14C cleaved from the This difference is highly significant (P < 0.001). The increased respective substrates was determined by liquid scintillation spec- glucocerebroside-cleaving enzyme activity in the leukemic leuko trometry according to the method described previously (7). cytes was not associated with an increased activity of the galacto- The enzymatic hydrolysis of sphingomyelin was determined on cerebroside-cleaving enzyme. 10- to 50-/il aliquots of the cholate extracts of sonicated leuko The mean level of activity of the sphingomyelin-cleaving en cyte preparations (14). The incubation mixtures contained 70 zyme was also higher in leukocytes from patients with acute and TABLE 1 Level of Sphingolipid Hydrolascs and Acid Phosphatase in Leukocyte Preparations from Normal Human Controls and Patients with Various Conditions PatientL.J.B.L.B.W.T.O.H.l).R.Y.R.S.J.K.R.R.P.J.F.S.E.N.Meanbrosidase5.03.44.94.74.74.15.54.34.04.74.95.64.6cerebrosidase5.32.94.03.84.23.12.23.92.73.63.6 melanomaParoxysmal nocturnalhe-moglobinuria Renal cell car cinomaHodgkin's dis easeGlucocere- ±S.E.Age(years)4138214434323031252827DiseaseNormalNormalNormalNormalNormalNormalNormalNormalMalignant±0.18Galacto-±0.28Sphingo-myelinase3.85.75.84.44.14.04.16.64.63.43.74.5±0.31Acidphosphatase3.54.25.44.23.35.04.85.24.14.9±0.24 The condition of incubation and assay procedure are described in the text. The values are ex pressed in m/imoles of substrate hydrolyzed per mg of protein per hour except for acid phosphatase which is Amólesper mg of protein per hour.