ABSTRACT
Since the induction of putrescine synthesis by ornithine decarboxylase (ODC) is observed in many pathological and physiological processes, a useful and simple method to assay this enzyme activity should be an interesting tool to quantify the biological importance of its induction. An enzymatic method to assay ODC is reported here. This method is based on the reaction between putrescine and soya diamine oxidase. The reaction releases H(2)O(2), which is measured by a colorimetric method. The validation of this method showed good accuracy (98+/-5% of recovery). High precision and reproducibility were obtained. A linearity with a correlation coefficient of 0.999 in the range of 2.5-25 nmol was obtained. This method is also rugged and specific. The application of the assay of ODC activity showed that it is useful as a rapid and simple tool for assaying ODC activity in vitro. Comparison with the HPLC determination of ODC activity shows strong correlation along with the high accuracy of the two methods.
ABSTRACT
Ifenprodil (NMDA receptor antagonist) was tested as an inhibitor of ornithine decarboxylase. It was found that ifenprodil inhibited ornithine decarboxylase activity with the same potency as alpha-difluoromethylornithine, a major inhibitor of ornithine decarboxylase. This result suggests that ifenprodil could target either the polyamine site on the NMDA receptor complex or/and polyamine biosynthesis.
Subject(s)
Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Ornithine Decarboxylase Inhibitors , Piperidines/pharmacology , Biogenic Polyamines/metabolism , Eflornithine/pharmacology , Escherichia coli/enzymology , Putrescine/analogs & derivatives , Putrescine/pharmacology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitorsABSTRACT
It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H2O2, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N1-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N1-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3-20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid (approximately 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity (approximately 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.
Subject(s)
Amebicides/toxicity , Amoeba/drug effects , Amoeba/enzymology , Oxidoreductases Acting on CH-NH Group Donors/physiology , Polyamines/toxicity , Animals , Cells, Cultured , Emetine/toxicity , Spermidine/toxicity , Spermine/toxicity , Toxicity Tests , Polyamine OxidaseABSTRACT
A simple reversed-phase HPLC method was developed for the determination of eight polyamines or monoacetylpolyamines, as their benzoylated derivatives. Interfering products, inherent to the benzoyl chloride derivatization technique (benzoic acid, methyl benzoate and benzoic anhydride), were identified. A new derivatization procedure for their total elimination was developed without any loss of sensitivity and selectivity. Not only the HPLC method was validated, but also the choice of an internal standard was investigated. The results show that it is possible to use this HPLC assay to determine the polyamine content in P388 cancer cells. Furthermore, the method is now being used to evaluate the uptake of various polyamines by P388 cancer cells and by other cancer and parasitic cells.
Subject(s)
Benzoates/analysis , Chromatography, High Pressure Liquid/methods , Polyamines/analysis , Animals , Benzoates/chemistry , Benzoic Acid , Kinetics , Leukemia P388 , Polyamines/chemistry , Reproducibility of Results , Solutions , Spectrophotometry, Ultraviolet , Tumor Cells, CulturedABSTRACT
A spectrophotometric method (cerium(III)-alizarin complexan-fluoride in presence of 25% dimethylsulfoxyde) is described for the determination of fluoride in human bones. The anion is determined after separation by microdiffusion as hydrofluoric acid using Petri boxes without any mineralization. This analytical method is selective, accurate and rapid.
