The detection and determination of polyamines from human urine [proceedings].

In recent years increased concentrations of the polyamines putrescine, spermidine and spermine have been reported in body fluids from humans and animals with a variety of malignant disorders. In addition, changes occurring in the spermidine/putrescine ratio in urine from cancer patients receiving chemotherapy appeared to provide an indication of the success or failure of the particular chemotherapeutic regime (Russell et al., 1971, 1975; Marton et al., 1973; Russell & Russell, 1975; Russell, 1977). To assess the prognostic capabilities of urinary polyamine determinations, it was first necessary to develop a routine assay procedure. Whereas Russell and her co-workers adapted an amino acid analyser to this end, Dreyfuss and his co-workers (Dreyfuss et al., 1973) and Seiler & Wiechmann (1 967) both described methods involving fluorescence quantification after t.1.c. separation of the dansyl* derivatives of the polyamines. Both these latter methods were subject to interference from urea and ammonia, and neither method allowed the separation of all three polyamines by a single chromatography system. We have therefore developed an assay based on the same underlying principles, but in which these problems have been overcome. Putrescine, spermidine and spermine were purchased from Sigma (London) Chemical Co. in the form of the hydrochloride salts. The aqueous standard solution contained 3mg of each polyamine, calculated as free base, per litre of solution. Dansyl chloride (Sigma) was dissolved in acetone to a concentration of 30mg/ml. Aspartic acid was dissolved in 1 . 5 ~ N a ~ C o ~ to a concentration of 100mg/ml. Na3PO4,I2Hz0 (21 g) and anhydrous Na,H04 (62.5g) (both of analytical-reagent grade) were ground together in a mortar and pestle to form the salt mixture. All other chemicals were of analytical-reagent grade. T.1.c. was carried out with silica-gel 60 HPTLC plates(Merck, Darmstadt, Germany) (10cm x 1Ocm). The chromatograms were developed in a single-plate Chromatank (Shandon Southern Ltd.). Specimens wereapplied to the t.1.c. plate with a 5 0 ~ 1 Hamilton syringe mounted in a Burkard hand micro-applicator. Quantification was achieved by using a Perkin-Elmer MPF-2A fluorescence spectrophotometer equipped with a thin-layer scanning attachment. A 3 ml sample of urine was mixed with an equal volume of conc. HCI in a Universal container and hydrolysed in an oven at 140°C for 16h. The hydrolysate was allowed to cool and 3 ml of 15 M-NaOH was added. The contents of the tube were heated at 3 0 T , and air was passed through the solution to remove NH3. The solution was made up to IOml with O ~ M N ~ O H , and 3g of saIt mixture added to give a solution of free bases (pH > 12). A 3 ml portion of the alkaline hydrolysate was then extracted with lOml of butan-1-01, and 8ml of the butanol layer was transferred to a separate tube. After addition of 100,d of conc. HCI, the butanol extract was evaporated to dryness. To the dried extract 0.5ml of 1 . 5 ~ N a , C 0 ~ was added, followed by an equal volume of dansyl chloride. The tube was sealed and left overnight in the dark. The large excess of dansyl chloride still remaining was removed by adding 0.5 ml of aspartate solution and leaving for 1 h in the dark. The dansyl-amines were then extracted into diethyl ether after addition of 2.5 ml of 0.5M-NaOH. The dansylaspartate remained in the aqueous phase during the extraction. A 2.5ml sample of the ether layer was then evaporated to dryness and the residue was redissolved in 2 0 0 ~ 1 of chloroform. A portion (5pl) of this solution was applied to the t.1.c. plate, and the plate was developed in a cyclohexane/ethyl acetate (3:2, v/v) solvent system to a height of 8cm. * Abbreviation: dansyl-, 5-dimethylaminonaphthalene1 -sulphonyl-.