Examination of nucleic acid-nucleotide metabolic processes has an outstanding importance in detecting tumorous processes and in distinguishing benignant and malignant processes from one another. Examination of the changes in nucleic acid metabolic processes is indispensible from the aspects of monitoring tumour therapy, too, since in the overwhelming majority of chemotherapeutical treatments the therapeutic effect appears in artificially provoked disturbances of the nucleic acid-nucleotide metabolism and in artificial damaging of DNA synthesis. These artificially provoked changes are decisive factors of the effectiveness of tumour therapy, however, they are also responsible for basic damages of healthy cells and disturbances of cell and organ functions.
Under routine conditions the determination of the activity of 5′-nucleotidase (5′-ribonucleotide phosphohydrolase), an enzyme detected by Reis (Reis, J.: Über die spezifische Phosphatase der Nerwengewebe: Enzymologia 2, 110-115/1937/), is one of the most appropriate methods. This enzyme catalyzes solely the hydrolysis of nucleotide pentose monophosphates (5′-AMP, 5′-CMP, 5′-UMP, 5′-GMP, 5′-IMOP, 5′-TMP, dAMP, dCMP, dUMP, etc.), yielding nucleotides and inorganic phosphate (Bodansky, O., Schwartz, M. K.: 5′-Nucleotidase; Adv. Clin. Chem. 11, 277-327/1968/), and can be regarded as one of the most sensitive plasm membrane markers. Determination of the activity of 5′-nucleotidase is used both for clinical purposes and in experimental tests; in the former instance measurment is performed usually on non-haemolysed serum and/or on a haemolysate, whereas in the latter instance measurement can be performed on body fluids, on tissue preparates (e.g. on plasm membranes) and/or on cell elements (e.g. on microsomes, cytoplasms, lysosomes) (see: Solyom, A., Trans., E. G.: Enzyme Markers in Characterization of Isolated Plasma Membranes: Enzyme 13, 329-372/1972/).
As 5′-nucleotidase is an enzyme which splits specifically nucleotide pentose monophosphates, a widely applied method for determining the activity of 5′-nucleotidase is to incubate the biological sample with a nucleotide pentose monophosphate substrate and then, after adding an appropriate colour developing reagent, to determine by colorimetry (spectrophotometry) the amount of inorganic phosphate liberated within unit time upon the effect of the enzyme. If the biological sample to be examined also comprises components which interfere with 5′-nucleotidase enzyme (for blood or serum such a component is alkaline phosphatase, for lysosome membrane such components are the other sugar phosphates), incubation is performed in the presence of an inhibitor capable of inhibiting their effects. As colour developing reagent ammonium molybdate is applied in the presence of a reducing agent, which is usually stannous chloride optionally together with a hydrazine salt orascorbic acid, which reacts with the inorganic phosphate to form an intense blue complex. Considering that ammonium molybdate precipitates the proteins present in the biological sample, proteins are either removed before performing the colour reaction, or proteins are kept in solution by adding an appropriate solubilizing agent to the mixture. To our recent knowledge, this latter is one of the most up-to-date calorimetric methods. Utilizing this method, the sample requirement of the determination can even be reduced to some hundredth milliliters, and the accuracy of the assay can be increased considerably. Determination of the activity of 5′-nucleotidase on the above principle is disclosed in detail, among others, in the following papers and in further references cited therein: J. Clin. Chem. Clin. Biochem. 7, 18-25 (1969), 13,453-459 (1975), 15, 715-718 (1977), 18, 781-788 (1980); Clin. Chem. 23, 2311-2323 (1977)127, 464-465 (1981); Adv. Clin. Chem. 11, 277-332 (1968).
A common feature of all routine methods applied before to determine the activity of 5′-nucleotidase on the above basis for diagnostic or general experimental purposes is that enzyme activity has been measured only on a single substrate. As substrate, most frequently adenosine-5′-monophosphate (5′-AMP), less frequently cytidine-5′-monophosphate (5′-CMP) or inositol-5′-monophosphate, or -in haemo-lysates-uridine-5′-monophosphate (5′-UMP) is applied.
USPTO Backgrounds
USPTO Backgrounds