THE IMPORTANCE OF ACETYLATION IN POLYAMINE METABOLISM AND EXCRETION (SPERMIDINE)

Abstract

To determine the structure(s) of the spermidine conjugate excreted into urine, extensive pharmacokinetic studies on the turnover of [¹⁴C]spermidine were conducted in both rats and humans. These studies demonstrated that exogenously administered [¹⁴C]spermidine equilibrated with endogenous polyamine pools. Radiolabeled urine collected from the humans was subjected to a cleanup protocol and subsequent analysis by GC-MS, which demonstrated the presence of N-acetylspermidine. Further analysis of the radiolabeled urine by thin layer chromatography (TLC) demonstrated the presence of both N¹- and N⁸-acetylspermidine in an approximate 1 to 1 ratio. Using similar methodologies the monoacetyl derivatives of putrescine and cadaverine were found to be the primary conjugated products of these polyamines excreted into human urine. Radiolabeled rat tissue extracts, analyzed by TLC, demonstrated that all tissues studied contained [¹⁴C] N¹- and N⁸-acetylspermidine. N¹-acetylspermidine was the primary isomer detected in all tissues; although, N⁸-acetylspermidine was detected in all tissues studied. The N-acetylspermidine content of an isolated cell system was determined in Chinese hamster ovary cells. These cells contained both N¹- and N⁸-acetylspermidine in an approximate 2 to 1 ratio. To directly measure the N-acetylpolyamines excreted into urine, two high performance liquid chromatography (HPLC) methods were developed. Both HPLC methods utilize a cation exchange resin, one using high pH, low salt buffers and the other low pH and high salt buffers. The primary N-acetylpolyamine excreted into human urine is N-acetylputrescine with lesser amounts of N¹- and N⁸-acetylspermidine which exist in a 1 to 1 ratio. In contrast, cancer patients excreted elevated amounts of both N-acetylputrescine and N¹-acetylspermidine. Cystic fibrosis patients were also found to excrete elevated amounts of N¹-acetylspermidine resulting in a consistently elevated N¹- to N⁸-acetylspermidine ratio. Mice injected with P-388 leukemia tumor excreted elevated amounts of N-acetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine. In contrast, the excretion of the unconjugated polyamines putrescine and spermidine in these animals was decreased, suggesting that the altered polyamine excretion was not primarily due to the presence of the tumor. Administration of Adriamycin to the tumor bearing animals resulted in the elevation of N¹-acetylspermidine excretion which was proportional to the relative tumor burden. Similar results were obtained from 2 human leukemia patients studied following chemotherapy.