Changes in Urine Polyamines in Childhood Leukemias*

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1 ANNALS O F CLINICAL AND LABORATORY SCIENCE, Vol. 11, No. 2 Copyright 1981, Institute for Clinical Science, Inc. Changes in Urine Polyamines in Childhood Leukemias* A. GARNICA, T. BENTON,f P. SLANINA,* T. MIALE, M.D., and A. L. STENKE" fgainesville, FL; \Uppsala, Sweden; Forth Worth, TX; and 1Stockholm, Sweden ABSTRACT Urine polyam ine: creatinine ratios (nm per mg) measured in eight children with active hematologic cancers were compared with those of agematched controls and children with hematologic cancer in remission. Polyam ine: creatinine ratios in the children with active disease were significantly higher than those of the controls (p < ) and of the children in remission (p < ). Putrescine: creatinine ratios were, in general, higher in children with hematologic tumors than in those with solid tumors. Urinary polyamines are thought to reflect variations in bone marrow polyamine content and have been postulated to be an indicator of clinical status. Introduction hematologic cancers. 1 1, 1 2, 1 4, 1 5, 2 8, 2 9 A return of polyamine excretion to normal has been postulated to reflect tumor regression in response to therapy, and the serial measurement of polyamine excretion to monitor the course of malignancy has been suggested. 1 2,2 7,2 9 Data on the urinary excretion of polyamines in childhood cancer is, however, limited. The present report documents the measurement of polyamines in unhydrolyzed urine samples from children with acute leukemia versus a variety of other childhood cancers. Patients with cancer in remission w ere included in order to correlate polyamine excretion with the clinical status of the disease. Urinary polyamines in humans occur as free forms and as acetyl conjugates but relatively little is known about pathologic conditions causing an increase in polyamine synthesis or excretion.4,1 2,1 6 Most reports reflect total (free plus conjugated) urinary polyamines measured after acid hydrolysis /81/ $00.90 Institute for Clinical Science, Inc. Rapidly growing or proliferating tissues, including human malignant tumors, are known to contain high concentrations of polyam ines. 6, 1 6, 1 7 As early as 1853, leukemic spleen was shown to be rich in spermine.8 However, the first successful application of polyamine determinations as indicators of malignancy was the demonstration by Russell et al of increased serum and urine polyamines in cancer patients.2 5,2 6 Rennert et al reported a correlation between bone marrow polyamine concentration and percentage of malignant cells present in the bone marrow in children with leukem ia. 2 1,2 4 Increased urinary excretion of polyamines has now been reported in patients with various malignancies but is most pronounced in * Supported in part by N IH G rant No. 1 K04 NS Requests for Reprints: A. Garnica, M.D., D ept, of Pediatrics, J-296, U niversity of Florida, Gainesville, FL

2 110 GARNICA, BENTON, SLANINA, M IALE, AND STENKE Materials and Methods P a t i e n t s a n d S p e c i m e n s Three groups of patients were studied. Eighteen patients had newly diagnosed, active cancer or were in relapse. The diagnoses included hematological malignancies (acute lymphoblastic leukemia [ALL], acute m yelogenous leukem ia [AML], chronic myelogenous leukemia [CML], lymphoma) and solid tumors (osteosarcoma, retinoblastoma, central nervous system tumor). In ten patients, the neoplastic process (ALL) was in remission. The mean ages of the two groups of patients were 12.4 and 11.9 years, respectively. Age-matched volunteers with no diagnosed malignancy served as controls. Both sexes were equally represented in the three groups. Patients were designated in complete remission following the criteria outlined by Fembach. 13 The patients w ere asym ptom atic; physical examinations were normal; peripheral blood studies and bone marrow morphology were normal when they were designated to be in remission. Anything short of full remission was designated active disease. Urine specimens and random (spot) urines were collected and refrigerated. Aliquot samples were frozen immediately after collection and stored without preservative at 20 C until analysis. For a comparison, approximately half of the specimens were divided and acidified with concentrated HC1 to ph 1 to 2 before freezing. An aliquot of each urine specim en was taken for creatinine determ ination. 9 A n a l y t i c a l M e t h o d s Cation-Exchange Analysis. Non-hydrolyzed urine specimens containing no protein were filtered through a Millipore filter (0.45 fim pore size) and 0.5 to 2.0 ml portions were lyophilized. The residue was reconstituted to one-fifth the original volume with deionized water and then centrifuged at 10,000 rpm. Forty m i croliters of the resulting supernatant were used for analysis. Hydrolyzed urine samples were prepared by the fluxing of urine with equal volumes of concentrated HC1 at 110 C for 16 hours. After acid hydrolysis, the samples were filtered and centrifuged. The supernatant was then collected and evaporated to dryness under a nitrogen-stream in a 70 C water bath. The residue was reconstituted with deionized water as described previously. Polyamine analysis was performed on an amino acid analyzer* with fluorometerf and modified as described by Marton and Lee. 18 Reagents: 0-pthalaldehyde 0.8 g was dissolved in 2 0 ml ethyl acetate: methanol (1:1). Twenty-five g of potassium hydroxide, 25 g of boric acid, and 5.8 g of potassium thiocyanate were dissolved in water and added to the OPA solution. Exactly 4.5 ml of 2-mercaptoethanol and 3.0 ml of 30 percent brij-35 were then added and the solution brought up to 1. 0 liter volume. Buffers: Buffers were prepared by the dilution of stock solutions containing 60 g of potassium citrate monohydrate, g of potassium chloride, and 1. 0 ml of liquified phenol per liter. Buffer 1 = 130:1000 dilution of stock buffer ([K+] = 0.42M). Buffer 2 = 480:1000 dilution of stock buffer ([K+] = 1.55M). Buffer 3 = 680:1000 dilution of stock buffer ([K+] = 2.20M).Buffer 4 = 900:1000 dilution of stock buffer ([K+] = 2.91M). S t a t i s t i c a l T e s t s The chi-square test for statistical differences was used for analysis of the experim ental data, or w hen applicable, Student s t test. Results The ratio of nm of urinary polyamines to mg of creatinine has been used as an expression of polyamine excretion. The use * M odel D500, Dionex, Sunnyvale, CA. f American Instrum ent Co., Silver Springs, MD. { OPA, Sigma Chemical Co., St. Louis, MO.

3 U RINE POLYAM INES IN CHILD H O O D LEUKEM IA 111 fulness of creatinine as a reference compound for studying urinary excretion has been established. 1 0,14,2 9 With reference to polyamine concentration, Russell reported that serial 24 hour urine specimens from the same patient demonstrated remarkably consistent values when polyamine excretion was expressed per mg of creatinine. 29 A comparison of putrescine: creatinine ratios was made between 24 hour urine collections and random urine samples from normal controls, and no significant difference in the values determined for the two specimens could be dem onstrated. Thus, the polyam ine: creatinine ratio in random urine specimens can be used as a measure of excretion. The effect of random day-to-day variation in polyamine concentration can be minimized when it is expressed per unit of urinary creatinine.29 All the unhydrolyzed urine specimens examined contained detectable concentrations of putrescine and spermidine. In some instances cadaverine and spermine were also detected in the urine of control and cancer patients. Hydrolysis of the specim ens resu lted in significant in creases in measurable spermidine and putrescine. The control value for polyam ine excretion (mean ± 2SD, nmoles per mg creatinine) was 1.61 ± for putrescine and 0.40 ± 0.20 for spermidine (table I, figure 1). The patients with active hematologic malignancies excreted significantly higher quantities of putrescine (p < ) and spermidine (p < ) as compared with the controls and the patients in remission (figure 1 ). Seven of eight patients w ith active hem atological m alignancies dem onstrated values for putrescine and spermidine excretion higher than either the normal control patients or those with hematologic malignancies in remission. The levels of putrescine and spermidine in the urine of patients with cancer in remission were not significantly different from those in control group (figure 1 ), all individual levels being within the normal range. Spermine levels were determ ined Active Cancer; Hematologic Non-Hematologic Remission, Hematologic o Normal Control A F ig u r e 1. U rinary p u trescin e and sperm idine in pediatric cancer patients. but are not included in this report because they demonstrated no correlation with clinical status. D ecreases in p u trescin e and sp erm idine could be dem onstrated when patients entered com plete remission. Although detailed serial studies were not systemically obtained, several patients had serial determ inations. G enerally markedly elevated values were found with active disease as opposed to normal values when patients obtained remission status. T A B L E I Urinary PolyamineiCreatinine Ratios (nm per mg) in Pediatric Cancer Patients Patient Group Putrescine Spermidine Hematologic, active *13.87 ± *2.67 ± 1.61 Hematolog ic, remi s s ion 1.13 ± ± 0.10 Non~hematologic 7.71 ± 6: ± Control 1.61 ± ± 0.10 *P <

112 GARNICA, BEN TO N, SLANINA, M IALE, AND STENKE Discussion Increased serum levels and increased urinary excretion of polyamines in patients with hematologic malignancies have been reported by R

4 112 GARNICA, BEN TO N, SLANINA, M IALE, AND STENKE Discussion Increased serum levels and increased urinary excretion of polyamines in patients with hematologic malignancies have been reported by R ussell et a 25,26,27,28,29 j n 0 g untreated patients, they recorded a mean urinary excretion of putrescine and spermidine two to three tim es that of norm al controls. 2 9,3 0 Moreover, increased putrescine concentrations have been recorded in the serum and urine of patients with various types of cancer. 1 4,2 2,3 2 Fujita et al found that total urine polyamines, especially putrescine, were increased and of diagnostic significance in patients with hematologic cancers. 14 They reported a significantly greater increase in urinary putrescine in patients with hematologic cancers as compared with solid tumors. Tsuji et al reported a marked increased in urine polyamines in patients with hematologic cancers but not solid cancers.32 A comparison of the levels of free putrescine and spermidine in random and 24 hour urine samples from children with active cancer versus age-matched controls and a group of patients in remission demonstrated significantly elevated levels in the active cancer group. These findings are sim ilar to those reported in hydrolyzed urine of adult cancer patients. 31 In this study, the highest elevations of free polyamines were observed in hematological malignancies, while other types of cancer show ed varying elevations of polyamines. These findings also correspond with the previously published results of hydrolyzed urine from adult cancer patients. 1 5,1 9,3 1,3 4 The high urine polyamine values observed in the patients with leukemia possibly reflect the elevated bone marrow polyamine levels reported by Rennert et al.24 According to several studies, the remission of a neoplastic process is associated ultim ately w ith a return of urinary polyam ine excretion to the normal range. 7,1 5,2 9 None of the patients in our remission group was found to have excessive polyamine excretion. The values observed in this group are perhaps the more significant in that all our cases were leukemic disorders. This result is consistent with findings previously reported by Miale et al, who related bone marrow putrescine levels to the remission status of children under treatment for leukemia and suggested a potential use for this m easurem ent prediction of relapse. 21 Rennert et al, moreover, reported cyclic variations in bone marrow levels of polyamines in leukemia patients over a period of several months, corresponding with changes in bone marrow cellularity. 2 1,2 4 In a group of children with leukemia, they found the bone marrow polyamine concentrations to correlate with cellularity and percentage of malignant cells in the bone marrow. Rennert et al also reported a sharp increase in bone marrow putrescine in two patients with leukemia several weeks prior to the clinical diagnosis of relapse by bone marrow aspiration.24 Thus, Miale et al suggested that serial measurements of bone marrow polyamine concentrations in leukemic patients might be utilized in the assessment of disease status and contribute to the early detection of relapse before the full-blown manifestations become apparent.21 It has been suggested that the increased quantities of polyamines in the extracellular fluids of cancer patients are derived primarily from tumor cells and might, therefore, serve as indicators of tumor kinetics. 1,2 7 The increased concentrations of polyamines in cancer patients have been attributed to a combination of tumor proliferation and cell death. 7, 1 5 Increased putrescine excretion is postulated to reflect the growth fraction of neoplastic tissue, w hile increased sperm idine and spermine are thought to be indicative of cell d estru ctio n and release of polyamines.2 7 Animal tumor transplant studies by Anderssen et al have shown an increase in polyamine levels and in tumor-

URINE POLYAMINES IN CH ILD H O O D LEUKEMIA 113 derived lactate dehydrogenase activity in cell-free ascites fluid during the regression of mouse Ehrlich tumor transplanted into Mongolian gerbils.

5 URINE POLYAMINES IN CH ILD H O O D LEUKEMIA 113 derived lactate dehydrogenase activity in cell-free ascites fluid during the regression of mouse Ehrlich tumor transplanted into Mongolian gerbils.2 Clinical observations have demonstrated two polyamine responses to successful therapy. 1 5,2 7,2 9 In this study, random urine specimens and 24 hour urine collections were used. The urinary polyamine values measured were quantitated in relation to urine creatinine in order to correct for day-today variations in urine concentrations.29 Polyamine: creatinine values in 24 hour urine specimens from normal children did not differ significantly from values obtained from random specimens.22 A study of serial random urines in two adults and three children demonstrated differences in urinary polyamine: creatinine ratios of normal subjects during a 24 hour period, but the differences were far smaller than the differences betw een norm al and cancer patients. Similar results have been reported by others who found no major diurnal variation in free or total polyamines in normal human urine.4,3 6 The use of urine polyamine excretion has been suggested as a diagnostic tool for cancer detection or as a means for assessing response to therapy. 1 5, 1 6, 2 6,2 7,2 9 However, it should be recognized that the majority of reports have dealt with the monitoring of patients with advanced cancer, rather than with the detection of cancer. Moreover, increases in urinary polyamines are not specific for malignancies. In individual patients, the effects of certain drugs or associated medical complications must be assessed. In studies of patients with non-malignant disease receiving specific drug therapy, increased urinary polyamine excretion was found in 30 to 40 percent. 10,32 Increased urinary polyamines are also commonly found in association with infectious processes and have been reported w ith pernicious anemia, hemolytic anemia, polymyositis, pulmonary tuberculosis, and psoriasis. 12 In conclusion, the results of the present investigation imply that the measurement of free urinary polyamines may afford a simple and specific method for following disease activity and predicting relapse in childhood leukemias. Close follow-up studies on larger numbers of individual cases as well as an assessment of the role of the various non-neoplastic diseases will be needed before the full clinical evaluation can be made. References 1. An d e r s s e n, G. and H e b y, O.: Kinetics of cell proliferation and polyamine synthesis during E hrlich ascites tum or growth. C ancer Res. 37: , An d e r s s e n, G., Re n g t s s o n, G., A l b in s s o n, A., R o s e n, S., and H e b y, O.: In c re a se d polyam ine concentration and tum or-derived lactate dehydrogenase activity in physiologic fluids during regression of a heterologous tum or transplant. Cancer Res. 38: , A b d e l-m o n e m, M. M., O h n o, K., N e w to n, N. E., and WEEKS, Ch.E.: Thin-layer chromatography and high pressure liquid chromatography o f th e dansyl p olyam ines. A dvances in Polyamine Research. Campbell R. A., et al, eds. New York, Raven Press, 1978, pp A b d e l -M o n e m, M. M. and O h n o, K.: P olyam ine m etabolism III. U rinary acetyl polyamines in human cancer. J. Pharmaceut. Sci. 67: , B r e m m e r, H. J., Ko h n e, L., and E n d r e s, W.: The excretion of diam ines in hum an urine. II. Cacaverine, putrescine, 1,3-diaminopropane, 2,2 -Dithiobis (ethylamine) and sperm idine in urine of patients w ith cystinuria and cystinlysinuria. Clin. Chim. Acta 32: , Ba c h r a c h, U.: Polyamines as chemical markers of malignancy. Ital. J. Biochem. 25:77-93, COHEN, S. S.: Conference on polyam ines and cancer. Cancer Res. 39: , C h a r c o t, J. M. and Ro b in, C. P.: Observation de leucocythemic. Compt Rendu Seances. Soc. Biol. Fil. 5:44-50, C h a s s o n, A. I., G rady, H. T., and St a n l e y, M. A.: Determ ination of creatinine by means of autom atic chem ical analysis. Amer. J. Clin. Path. 35:83-88, D r e i l in g, D. A., L e i c h t l i n g, J. J., and J a n o w it z, H. D.: T he am ylase-creatinine clearance ratio. Amer. J. Gastroentro. 61: , D r e y f u s s, F., C h a y e n, R., D r e y f u s s, G., D v ir, R., and Ra t a n, J.: Polyam ine excretion in th e u rin e o f cancer patients. Israel J. M ed. Sci. 11-, , D u r ie, B. G. M., Sa l m o n, S. E., and Ru s s e l l, D. H.: Polyamines as markers of response and d isease activ ity in can cer chem o th erap y. Cancer Res. 37: , 1977.

114 GARNICA, BEN TO N, SLANINA, M IALE, AND STENKE 13. F e r n b a c h, D. J.: N atural history of acute leukemia. Clinical Pediatric Oncology, 2nd ed. Suton, W. W., Vietti, T. J., and Fernbach, D. J., eds.

6 114 GARNICA, BEN TO N, SLANINA, M IALE, AND STENKE 13. F e r n b a c h, D. J.: N atural history of acute leukemia. Clinical Pediatric Oncology, 2nd ed. Suton, W. W., Vietti, T. J., and Fernbach, D. J., eds. St. Louis, C. V. Mosby Co., 1977, pp F u jita, K., N a g a ts u, T., M u r u t a, K., I o to, M., S e n b a, H., and Mik i, K.: Urinary sperm idine and spermine in hum an blood and solid cancers and in an experimental gastric tum or in rats. Cancer Res. 36: , JANNE, J., Poso, H., and R a in a, A.: Polyamines in rapid growth and cancer. Biochim. Biophys. Acta 473: , L ip to n, A., S h e e h a n, L. M., and K e s s le r, G. F.: Urinary polyamine levels in hum an cancer. Cancer 35: , M a n d s le y, D. V.: Regulation of polyamine biosynthesis. Biochem. Pharmac. 28: , M a r t o n, L. G. and Le e, P. L. Y.: More sensitive autom ated detection of polyam ines in physiological fluids and tissue extracts with O-phtaladehyde. Clin. Chem. 2 1 : , M a r t o n, L. J., V a u g h n, J. G., H a w k, I. A., L ev y, C. C., and R u s s e l l, D. H.: Elevated polyamine levels in serum and urine of cancer p a tie n ts. D etectio n by rap id au to m ated technique utilizing an amino acid analyzer. Polyamines in Normal and Neoplastic Growth. Russel, D. H., ed. New York, Raven Press, 1978, pp M a ts u d a, M., O s a f u n e, M., K o ta k e, T., S o n a d a, T., S o b u e, K., and NAKAJIMA, T.: Concentrations of polyam ines in renal cell carcinomas. Clin. Chim. Acta 87:93-99, M ia le, T. D., R e n n e r t, O. M., L a w so n, D. L., S h u k la, J. B., and F r ia s, J. L.: Bone marrow polyamines in children with acute leukem ia as related to remission status, therapy and cellularity of specimens. Med. Ped. Oncol. 3: , M i a le, T. D., B e n t o n, T., S l a n i n a, P., S t e n k e, L., and G a r n i c a, A. D.: Polyam ine:creatinine ratios in crude urine samples of pediatric cancer patients (submitted). 23. NESHIOKA, K. and ROMSDAHL, M.: Elevation of putrescine and spermidine in sera of patients w ith solid tumors. Clin. Chim. Acta 57: , R e n n e r t, O. M., M ia le, T. D., S h u k la, J. B., L a w so n, D. L., and F r ia s, J. L.: Polyamine concentrations in bone marrow aspirates of children w ith leukem ia and other malignancies. Blood 47: , Ru ssel l, D. H.: Increased polyam ine concentrations in urine of hum an cancer. Nature New Biol. 232: , R u s s e l l, D. H., L ev y, C. C., Sc h im p f f, C. C., and H a w k, I. A.: U rinary polyam ines in cancer patients. C ancer Res. 37: , Ru s s e l l, D., D u r ie, B. G., and Sa l m o n, S. E.: Polyamines as predictors of success and failure in cancer chem otherapy. L ancet 2: , RUSSELL, D. H.: Polyam ines in grow th normal and neoplastic. Polyamines in Normal and Neoplastic Growth. Russell, D. H., ed. New York, Raven Press, 1978, pp Ru s s e l l, D. H.: C lin ical reiev an ce of polyamines as biochem ical markers of tumor kinetics. Clin. Chem. 23:22-27, T a b o r, H., T a b o r, C. W., and I r r e v e r r e, F.: q u a n tita tiv e d e te rm in a tio n o f alip h atic diam ines and polyam ines by an autom ated liquid chromatography procedure. J. Biochem. 55: , T o w n s e n d, R. M., Ba n d a, P. W., and M a r t o n, J. L.: Polyam ines in m alignant m elanom a. Cancer 38: , Tsujl, M., NAKAJIMA, T., and S a n o, I.: Putrescine, sperm idine, N -acetylsperm idine and sp erm in e in th e u rin e of p a tie n ts w ith leu k em ias and tum ors. C lin. C him. Acta 59: , Vil l a n u e v a, V. R. and Ak la k h a, R. C.: Automated analysis of comon basic amino acids, mono-, di- and polyamines, phenolic amines and indoleamines in crude biological samples. Analyt. Biochem. 91: , W a a l k e s, T. P., G e h r k e, C. W., Bl e y e r, W. A., Z u m w a l t, R. W., O l w e n y, C. L. M., Ku o, K. C., La k in g s, D. B., and Ja c o b s, S. A.: Potential biological markers in Burkitts lymphoma. Cancer Chemother. Rep. 59: , W a a l k e s, T. P., G e h r k e, C. W., T o r m e y, D. C., Z u m w a l t, R. W., H u e s s e r, J. N., Kuo, K. C., La k in g s, D. B., Ah m a n n, D. L., and M o e r - TEL, Ch. G.: Urinary excretion of polyamines by patients w ith advanced m alignancy. Cancer Chemother. Rep. 59: , Wa l l e, T.:. Gas chromatography mass spectrometry of di- and polyamines in human urine: Identification of m onoacetylsperm idine as a major metabolic product in sperm idine in a patie n t w ith acute m yelocytic leukem ia. Polyamines in Normal and Neoplastic States. Russell, D. H., ed. New York, Raven Press, 1978, pp