Enzymatic method for selective determination of acetylpolyamine as a biomarker
Kanto Gakuin University, Yokohama, Japan
AIM: Since Russel et al. reported that the amount of polyamine (PA) in urine of cancer patients was significantly larger than that in normal urine, urine PA has been attracting attention as a tumor marker of organ-nonspecificity for screening of cancer patients etc. It comprises acetylPA (AcPA) and non-acetylated PA (free PA), and clinically significant PA has been reported to be AcPA. The determination of AcPA is performed with special instrumental analysis, e.g. high performance liquid chromatography, which requires expensive instruments and complicated and tedious operation. As for the enzymatic methods, they have been limited to determine total PA without distinction between AcPA and free PA. The authors previously reported an enzymatic method for the selective determination of AcPA. However, its detecting sensitivity was only just enough for practical use. Here, the authors present a new convenient enzymatic method of high sensitivity for the determination of AcPA itself. METHODS: The method was based on five consecutive enzymatic reactions coupled with five enzymes, respectively, acylpolyamine amidohydrolase, acetate kinase, pyruvate kinase, pyruvate dehydrogenase and diaphorase; first, AcPA was hydrolyzed to yield acetate; finally, acetate liberation caused formation of diformazan as an indicator of the reaction. The amount of AcPA was then evaluated as the increase in absorbance at 550 nm due to the diformazan. Acetylputrescine (AcPut) was used as a typical AcPA in urine. After addition of AcPut to the reaction mixture, the increase in absorbance at 550 nm was measured within 15 min at 30°C. RESULTS: The reagent composition of the reaction mixture was determined, and characteristics of the method were investigated. A linear relationship existed over a wide range of concentration from 0 to 500 µM of AcPut. Within- and between-day precision tests produced satisfactory results, i.e. CV values were 1-2 %. The limit of detection reached a trace amount of AcPut as ca. 4 µM. Eight kinds of substances that may usually coexist in urine, including non-acetylated putrescine (free Put), gave no effect on the measurement. CONCLUSIONS: The present method proved to be successful in the selective and sensitive determination of AcPA alone even when free PA coexisted. Moreover, it is noteworthy that highly reliable results were easily and rapidly obtained through this method.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 893 (Molecular pathology).