35, 322-326 (1986)
Increased Urinary Polyamine Excretion during Liver Regeneration SIW ANEHUS,”
AND OLL.E HEW**’
*Department of Zoophysiology, University of Lund, Helgonavdgen 3, S-223 62 Lund; f&?partment of Surgery, University of Lund, S-221 85 Lund; and $Department of Surgery1, Sahtgrenska sjukhuset, University of Giiteborg, S-413 45 Giiteborg, Sweden Received March 8, 1985
The concentrations of the polyamines putrescine, spermidine, and spermine are elevated in physiological fluids of patients harboring a wide variety of solid and hematological tumors (1). In fact, quantitative analysis of physiological fluid polyamines may serve as a diagnostic aid in conjunction with established techniques and may provide valuable information regarding tumor progression and regression. Accordingly, Marton et al., (2,3) have demonstrated that elevated levels of cerebrospinal fluid polyamines are predictive of recurrence of human medulloblastomas. It has been proposed that the level of putrescine in physiological fluids reflects the proliferative activity of the tumor, while the level of spermidine reflects the incidence of tumor cell death (4,5). Results obtained in our laboratory, however, indicate that cell death is associated with elevated levels of putrescine rather than spermidine (6,7). In agreement with this notion, Ccl, intoxication of rats caused an increased excretion of putrescine, which coincided with the period of maximum liver damage (8). It cannot be entirely excluded, however, that even proliferating cells, with their rapid polyamine synthesis and accumulation, release polyamines at an elevated rate and thus contribute to increased urinary excretion of polyamines. To further analyze this possibility, the urinary polyamine excretion was determined during liver regeneration (after partial hepatectomy), a proliferative event associated with minimal cell necrosis. METHODS Animals and partial hepatectomy. Young male Wistar rats weighing 170-200 g were used. They were provided with standard laboratory feed and tap water, and were allowed to eat and drink ad libitum. Partial hepatectomy was carried out according to the procedure of Higgins and Andersson (9). Thus, approximately 70% of the liver mass was removed. All efforts were made to minimize the amount of hepatic tissue distal to the site of ligation. Sham operations were ’ To whom requests for reprints should be addressed. 322 08854505/86 $3 .OO Copyright All rights
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
carried out during comparable anesthesia and duration of operation. The operations were carried out between 8 and 10 AM. Collection of urine. To avoid bacterial contamination, 24-hr urines were collected in acid (0.5 ml 6 M HCl; sufficient to maintain an acidic pH) in metabolic cages (Econo Model E-1100 metabolism units, Maryland Plastics, N.Y.) modified to minimize evaporation. The urine samples were collected for 9 consecutive days from seven rats maintained in individual cages. The 24-hr urine volumes were measured at the same time each morning. Aliquots of the urine samples were centrifuged at 1000 g for 10 min and the supernatants were stored at -20°C until analysis. Determination of urinary polyamines. The total amount of free and bound (e.g., acetylated) polyamines was determined in I .OO-ml aliquots of the 24-hr urine collections after acid hydrolysis (6 M HCl; 14-16 hr; 1lO’C). After neutralization, 200-~.~1samples were analyzed for their polyamine content by a thinlayer chromatographic method (10) with the modifications previously described (11). RESULTS Urine volume. The 24-hr urine volume reached a maximum level (27 ml) during the first day after partial hepatectomy (Fig. 1). The urinary output then decreased steadily and was close to basal level (8.5 ml) by Day 6 after operation. Sham operation had no significant effect on the 24-hr urine volume. Urinary polyamine excretion. The 24-hr putrescine excretion increased during the first 2 days after partial hepatectomy (Fig. 2A). By Day 2, the level was more than two-fold higher than the basal level. Since the polyamine excretion varied among rats, the experimental data are expressed as percentage of the Day 0 value. From Day 2-4 after operation, the putrescine excretion decreased by about 60% of the peak value. After Day 4, however, there was no further decrease. 30‘= Jz .:: 20z s H o .5 3
lo_ OL ’ -2
2 Days after partial hepatectomy
FIG. 1. Twenty-four hour urine volume at various times after partial hepatectomy (means 2 SEM). Partially hepatectomized rats (0) (n = 4); sham-operated rats (0) (n = 3).
Days after partial hepatectomy
FIG. 2. Twenty-four hour urinary excretion of putrescine (A) and spermidine (B) at various times after partial hepatectomy (means f SEM). Partially hepatectomized rats (0) (n = 4); sham-operated rats (0) (n = 3). One hundred percent (Day 0) corresponds to 2.42 * 0.58 and 2.07 f 0.87 pmole of putrescine per 24 hr in rats subjected to partial hepatectomy and sham operation, respectively; and 1.05 + 0.18 and 1.21 +- 0.15 pmole of spermidine per 24 hr in rats subjected to partial hepatectomy and sham operation, respectively.
No changes in the urinary spermidine excretion were revealed during the first day after partial hepatectomy (Fig. 2B). During the second day, the 24hr spermidine excretion increased and a level almost two-fold higher than the basal level was reached. The spermidine excretion remained at this elevated level throughout the experiment. Sham operation exerted no significant effect on the 24-hr urinary putrescine excretion, but the 24-hr spermidine output increased steadily from Day 2 after sham operation and was 35% higher than the basal level by Day 6. Spermine was barely detectable in the urines and was not recorded. DISCUSSION
The observed increases in urinary putrescine and spermidine excretion in partially hepatectomized rats (Fig. 2) are preceded by increased synthesis and accumulation of putrescine and spermidine in the regenerating liver (12,13). The hepatic putrescine concentration reaches its maximum level during the first day and the subsequent decrease coincides with maximum rate of urinary putrescine
excretion. The maximum level of hepatic spermidine concentration is also reached during the first day and is followed by an increased rate of urinary spermidine excretion. The rates of polyamine synthesis and excretion remain elevated during the remainder of the regeneration process. The increase in urinary polyamine excretion may at least partly emanate from the increased synthesis of polyamines in the regenerating liver. However, there is no apparent explanation for the fact that the hepatic concentration of spermidine is IO-fold higher than that of putrescine, whereas the amount of spermidine excreted in the urine is only half that of putrescine (cf. Fig. 2 and Ref. 13). This difference may be due to a greater release of putrescine than of spermidine from the liver cells. In partially hepatectomized rats, the urinary putrescine excretion reaches a level that is only about 50% of that observed during CC&-induced liver regeneration (8). This lower rate of excretion may be explained by the fact that the liver mass is reduced by approximately 70% at operation. Despite the extensive reduction in liver mass, the putrescine excretion increases already during the first day. Conceivably, intraperitoneal absorption of polyamines released due to necrosis of the small amount of hepatic tissue that remains distal to the site of ligation may contribute to this early increase in urinary putrescine. The increase in urinary putrescine and spermidine excretion coincides with the maximum rate of DNA synthesis during liver regeneration (cf. Fig. 2 and Ref. 13). However, it cannot be generally assumed that proliferating cells release putrescine and spermidine at a higher rate than do nonproliferating cells. The observed increase in the excretion of these polyamines may be partly due to cell damage or permeability changes in the cell membranes during the initial phase of liver regeneration. This idea is supported by analyses which show that liver enzymes are released into the circulation during the course of regeneration (14). Thus, the plasma or serum activities of lactic dehydrogenase (14,15), yglutamyl transpeptidase, glutamic oxaloacetate transaminase, and alkaline phosphatase (14) increase and reach peak activities within 2 days of partial hepatectomy . Although there is an apparent correlation between elevated urinary polyamine excretion and the proliferative activity in the liver, concurrent permeability changes and necrotic events (which cannot be easily controlled for) complicate the interpretation of this relationship. Therefore, it still remains to be established to what extent the increased urinary excretion of polyamines, observed in association with normal or neoplastic growth, is due to release from rapidly proliferating cells. SUMMARY
Tumor growth is a process associated with both cell proliferation and cell death. The increase in polyamine excretion observed in cancer patients may be partly due to leakage of polyamines from proliferating cells, which all contain an elevated polyamine level. However, the increased polyamine excretion may also be due to a release of polyamines from dead or damaged cells. To determine if actively proliferating cells release polyamines, the urinary polyamine excretion was measured during a proliferative event associated with minimal cell necrosis. Rats subjected to partial hepatectomy were used as an experimental model. Their
24-hr urines were collected during 6 consecutive days following the operation. Rat liver regeneration is characterized by a proliferation wave with a maximum 24 hr after the operation. The 24-hr urinary putrescine excretion reached a maximum 2 days after the operation and then decreased. The 24-hr urinary spermidine excretion increased during the second day following operation and remained essentially unchanged during the rest of the experimental period. Although there is an apparent correlation between elevated urinary polyamine excretion and the proliferative activity, concurrent permeability changes and necrotic events may contribute to the increase in polyamine excretion. ACKNOWLEDGMENTS This investigation was supported by grants from the Swedish Natural Science Research Council, the Medical Faculty (University of Lund), the Segerfalk Foundation, the John and Augusta Persson Foundation, and the Royal Physiographical Society, Lund. We thank Marianne Andersson for invaluable help with drawings and typing.
REFERENCES 1. Marton, L. J., and Seidenfeld, J., in “Polyamines in Biology and Medicine” (D. R. Morris and L. J. Marton, Eds.), p. 337. Dekker, New York, 1981. 2. Marton, L. J., Edwards, M. S., Levin, V. A., Lubich, W. P., and Wilson, C. B., Cancer Res. 39, 993 (1979). 3. Marton, L. J., Edwards, M. S., Levin, V. A., Lubich, W. P., and Wilson, C. B., Cancer 47, 757 (1981). 4. Russell, D. H., Durie, B. G. M., and Salmon, S. E., Lance?, 2, 797 (1975). 5. Durie, B. G. M., Salmon, S. E., and Russell, D. H., Cancer Res. 37, 214 (1977). 6. Heby, O., and Andersson, G., Acta Pathol. Microbial. Scand. Sect. A 86, 17 (1978). 7. Andersson, G., Bengtsson, G., Albinsson, A., RosCn, S., and Heby, O., Cancer Res. 38, 3938 (1978). 8. Anehus, S., Yngner, T., Engelbrecht, C., Hafstriim, L., and Heby, O., Exp. Mol. Pathol. 38, 255 (1983). 9. Higgins, G. M., and Andersson, R. M., Arch. Pathol. 12, 186 (1931). 10. Seiler, N., Methods Biochem. Anal. 18, 259 (1970). 11. Anehus, S., Bengtsson, G., Andersson, G., Carlsson, G., Hafstriim, L., Yngner, T., and Heby, O., Eur. .I. Cancer 17, 511 (1981). 12. Dykstra, W. G., Jr., and Herbst, E. J., Science 149, 428 (1965). 13. Piis& H., and Pegg, A. E., Biochim. Biophys. Acia 696, 179 (1982). 14. Sekas, G., and Cook, R. T., Brir. J. Exp. Parhol. 60, 447 (1979). 15. Yngner, T., Bengtsson, G., Carlberg, E., Engelbrecht, C., and Wieslander, A., Exp. Cell Biol. 48, 393 (1980).