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Urinary Putrescine, Spermidine, and Spermine in Human Blood and Solid Cancers and in an Experimental Gastric Tumor of Rats

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Urinary Putrescine, Spermidine, and Spermine in Human Blood and Solid Cancers and in an Experimental Gastric Tumor of Rats (CANCER RESEARCH 36, 1320-1324, April 1976] Urinary Putrescine, Spermidine, and Spermine in Human Blood and Solid Cancers and in an Experimental Gastric Tumor of Rats Kelsuke Fujita,1 Toshiharu Nagatsu, Kazuhiro Maruta, Madoka Ito, Hideo Senba, and Kazuki Miki Institute for Comprehensive Medical Science, Fukita-Gakuen University School of Medicine, Toyoake, Aichi 470-11, Japan (K. F., K. M., M. I., H. S., K. MI, and Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Nagoya 464, Japan (T. N.J SUMMARY missions were observed. The patients had normal renal function, and patients with nephrosis or nephritis were not An improved method of assay of urinary polyamines (pu included, because it has been observed that urinary polya trescine, spermidine, and spermine) was applied to the mine concentrations rose in some but not all of the patients study of cancer patients and an experimental gastric tumor with nephrosis (K. Fujita, T. Nagatsu, K. Maruta, and M. Ito, of rats. Although total polyamines (putrescine, spermidine, unpublished results). and spermine) in urine of patients with blood and solid Experimental Stomach Cancer. For animal model studies cancers were significantly high , putrescine concentrations of tumors in the glandular stomach (6), 3.5 ml of NG2 also increased significantly and were shown to be of diag aqueous solution (2000 mg/liter) were administered to 20 nostic aid even in solid cancers. A significant increase in male Wistar rats through a stomach tube once a week for 42 putrescine was also noted in the urine of rats with experi weeks. Twenty control rats were given water freely. All rats mental stomach tumors induced by N-methyl-N-nitro-N' were sacrificed 300 days after continuous administration of nitrosoguanidine. a total dose of 249 mg of NG and were autopsied. The tumor incidence in the glandular stomach of 11 rats that survived INTRODUCTION after the administration of NG was 100%, whereas no tumor The polyamines (putrescine, spermidine, and spermine) was observed in the surviving 9 control rats. Twenty-four-hr are intimately associated with many aspects of cell growth, urine specimens were collected in a metabolism cage with a and since the first demonstration by Russell et a!. (3, 4) that urine collector with each rat. urinary polyamines increased in human cancer, the deter Determination of Polyamines (Putrescine, Spermidine, mination of polyamines in the urine of cancer patients has and Spermine) in 24-Hr Urine Samples. The original received increased attention. method of Russell et al. (4, 5) was modified by using a small We improved and simplified the assay method of Russell Dowex 50 column for the isolation of polyamines as devised et a!. (4, 5) by using column chromatography (2), and we by lnoue and Mizutani (2). Twenty-four-hr urine samples applied this method for determining urinary polyamines in were collected under toluene and refrigerated. A 5.0-mI patients with blood cancers and various solid cancers. Al aliquot of the 24-hr urine sample was hydrolyzed in 2 N HCI though our values of total urinary polyamines were similar for 3 hr at 100°.Ahigher concentration (6 N)of HCI or longer to those reported by Russell et a!. (3, 4), it was found that the heating (up to 16 hr) or higher temperature (110-120°)did increase of putrescine was more significant in patients with not increase the polyamine contents. After hydrolysis, the blood cancers as well as solid cancers. We also noted a sample was neutralized with 1 N NaOH and was applied to a significant elevation of urinary putrescine in experimental column of Dowex 50W-X8 (200 to 400 mesh) (9 x 40 mm). stomach tumor of rats. The column was washed with 60 ml of 0.1 M sodium phos phate buffer, pH 8.0, containing 0.7 M NaCI, and then with 60 ml of 0.5 N HCI. Polyamines were eluted with 15 ml of 6 N MATERIALS AND METHODS HCI, and the eluate was evaporated to dryness in a rotary evaporator at 60°.Theresidue was dissolved in 200 pJ of 0.1 Selection of PatIents for UrIne Collection. Twenty-four-hr N HCI. A 2O-pi aliquot of the sample was applied to a paper urine samples were collected from 56 normal subjects, 2 strip (15 x 57 cm, Whatman No. 3MM paper) previously pregnant women, 26 postpartum women, and 72 nontumor moistened with 0.07 M sodium citrate buffer, pH 4.3. High patients hospitalized for a variety of diseases. The cancer voltage paper electrophoresis was carried out at 5°for 10 patients admitted to the Fujita-Gakuen University Hospitals mm at 80 V/cm, using a Servant electrophoresis chamber. had blood or solid cancers. Urine samples were cpllected As standard, 10 @lofa polyamine mixture containing 5 mM before initiation of chemotherapy, radiation therapy, and putrescine, 2.5 mM spermidine, and 2.5 mM spermine were surgery for the removal of a portion of a solid tumor mass; simultaneously subjected to electrophoresis. After electro additional samples were collected after treatment when re phoresis, the paper strip was dried at 70°for15 mm and was I Towhomrequestsforreprintsshouldbeaddressed. Received September 19, 1975; accepted December 10, 1975. 2 The abbreviation used is: NG, N-methyl-N-nitro-N'-nitrosoguanidine. 1320 CANCER RESEARCH VOL. 36 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1976 American Association for Cancer Research. Urinary Polyamine Assay in Humans and Rats sprayed with ninhydrin reagent containing 1 g of ninhydrin, pared with those of untreated cancer patients (Table 1), 0.1 g of cadmium acetate, 10 ml of water, 5 ml of acetic acid, total polyamines were significantly reduced. Again, the re and 100 ml of acetone. After having been heated at 80°for1 duction in urinary putrescine levels was the most signifi mm to locate the polyamines, the ninhydrin solution was cant. In contrast, urinary spermidine levels showed a tend applied on the paper with a paint brush and the color was ency to remain elevated. Comparison was made (Table 3) fully developed by heating further at 80°for5 mm. The spots between the urinary polyamines (mg/24 hr) in the present of polyamines were directly measured with a Shimadzu CS report and those described in the report by Russell e! a!. (4). 900 dual-wavelength thin-layer chromatography scanner at Total polyamines were very similar to those reported by 505 and 700 nm by the zigzag scanning method. All the Russell et a!. (3, 4), but the elevation of urinary putrescine assay procedure can be finished within 7 hr. Recoveries of level in patients with blood cancers and solid cancers was [‘4C]polyaminesadded to urine samples were determined more pronounced in our study. with the samples before the high-voltage paper electropho To confirm the significant elevation of putrescine in solid resis. Urinary creatinine concentrations were determined by cancers, changes in urinary polyamines were studied in the method of Bonsnes and Taussky (1). animal model studies. Rats were fed NG, as described in “MaterialsandMethods,―by the procedure of Sugimura and Fujimura (6). All 11 rats that survived after 42 weeks of RESULTS continuous administration had stomach cancer. As shown in Chart 2, putrescine, spermidine, and spermine increased The present assay method of urinary polyamines is a in the urine of rats after administration of NG, and the combination of the methods of Russell et a!. (4, 5) and lnoue urinary putrescine level was most significantly elevated and Mizutani (2). As with the results by lnoue and Mizutani, compared to that of control rats. A significant increase in the separation of polyamines by chromatography with urinary putrescine in experimental rats was observed as Dowex 50 gave high recoveries, was reproducible, and elim early as 20 weeks, and it became marked during the ad mated interfering ninhydrin-positive substances. The mean ministration of NG. recoveries of [‘4C]polyaminesadded to urine were 99 ± 0.2%. Furthermore, the dual-wavelength densitometry with the zigzag scanning method was simple, sensitive, and DISCUSSION more accurate than the previously used extraction and co lorimetry procedure (2, 5). As shown in Chart 1, the assay The assay procedure described here is reproducible, ac was highly reproducible. curate, and considerably simpler than the original butanol Urinary polyamines expressed as mg/24 hr or nmole/mg extraction-electrophoresis procedure of Russell et a!. (3, 4). creatinine in normal subjects, nontumor patients, and pa The Dowex 50 column procedure for the isolation of polya tients with cancers are shown in Table 1. The total polya mines (2) is superior in simplicity, constant recovery, and mine levels were significantly elevated in normal pregnancy, isolation of the purer polyamine fraction. High-voltage pa blood cancers, and some solid cancers. An important ob per electrophoresis at 4°canshorten the time of electropho servation in this work is a significant elevation of putrescine resis from 2 hr (4, 5) to 10 mm. Direct densitometry by dual in blood and solid cancers. As shown in Table 1, urinary wavelength photometry with the zigzag scanning method is putrescine levels of nontumor patients were not signifi also simpler and highly accurate compared to the extraction cantly different from those of normal subjects, but in almost of polyamines from the paper and subsequent colorimetry. all kinds of blood cancers and solid cancers they were Our results on increase of total urinary polyamines in significantly higher than in normal and nontumor subjects. normal pregnancy and blood and solid cancers agree with Urinary polyamine levels in patients with various cancers the results of Russell et a!. (3, 4). Recently, Tsuji et a!. (7) after effective treatments are shown in Table 2.
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