Quantitation of polyamines in cultured cells and tissue homogenates by reversed-phase high-performance liquid chromatography of their benzoyl derivatives.
A rapid and simple method, originally described by Redmond and Tseng [J. Chromatogr., 170 (1979) 479] was applied to the analysis of di- and polyamines in cultured human tumour cells and human tumour xenografts. Optimization of the procedures and evaluation of the characteristic features of the assay are described. The (modified) procedure employs precolumn derivatization with benzoyl chloride, extraction of the derivatives by chloroform, separation by reversed-phase high-performance liquid chromatography under isocratic conditions and detection by ultraviolet absorbance measurement at 229 nm. The complete analysis was accomplished within 10 min per sample. The detection limit was ca. 1 pmol. The intra- and inter-assay coefficients of variation were 2.5-4.4% and 3.4-13.1%, respectively. The presence of well known inhibitors of polyamine biosynthesis, such as DL-alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone), did not interfere with the assay, and disturbance by cyclohexylamine could be avoided by changing the polarity of the mobile phase. The method proved to be very suitable because it is rapid, simple, requires a minimum of sample pretreatment, and still provides sufficient sensitivity to quantitate polyamines in relatively small amounts of cells (10(5) cells) or tumour tissues (less than 1 mg), even after treatment with inhibitors of polyamine biosynthesis.
Verkoelen
C F
CF
Department of Urology, Erasmus University, Rotterdam, The Netherlands.
Romijn
J C
JC
Schroeder
F H
FH
van Schalkwijk
W P
WP
Splinter
T A
TA
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D011073:Polyamines / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D014407:Tumor Cells, Cultured / Q000032:analysis*
D011073:Polyamines