CS218345B1 - A method for determining the visible activity of thiol derivative activated creatine kinase in the visible spectrum - Google Patents

Abstract

The invention relates to a method for determining the catalytic activity of creatine kinase activated by thiol derivatives in the visible region of the spectrum. The essence of the invention lies in the fact that the excess of thiol activators, interfering with the reaction of the reduced form of nicotinamide adenine dinucleotide with a chromogen, is removed by adding N-ethylmaleimide in an amount of 2 to 4 moles per 1 mole of thiol activator, after which the chromogen is added and the resulting color is measured in the visible region of the spectrum. The use of the invention eliminates the need for photometry in the ultraviolet region of the spectrum in the determination of activated creatine kinase, and thus allows the extension of this method to smaller clinical biochemistry laboratories.

Description

The present invention provides a method for determining the catalytic activity of thiol derivative-activated creatine kinase in the visible region of the spectrum.

Creatine kinase is an enzyme important for the function of striated muscles. It catalyzes the transfer of the phosphate group between adenosine triphosphate and creatine phosphate. The enzyme enters the blood from damaged muscle cells. Determination of its catalytic activity in serum is of great importance in diseases affecting striated muscles, for example in progressive muscular dystrophy and in particular in myocardial infarction, where the monitoring of serum creatine kinase activity is not only diagnostic but also prognostic.

A complex of three successive reactions is used to determine the catalytic activity of creatine kinase, the first of which is catalysed by creatine kinase. In the auxiliary and indication reaction catalyzed by hexokinase and glucose-6-phosphate dehydrogenase enzymes, the first reaction product is then converted to form a reduced form of nicotinamide adenine dinucleotide.

The formation of this compound can be monitored photometrically in ultraviolet light as an increase in absorbance which is proportional to the catalytic activity of creatine kinase. The determination therefore requires a photometer with the possibility to measure in the ultraviolet range of the spectrum; this photometer is expensive and unavailable in smaller clinical biochemistry laboratories.

In the determination of the activity of other enzymes, especially dehydrogenases, which also produce a reduced form of nicotinamide adenine dinucleotide, it has been described to convert measurements from ultraviolet to the visible region of the spectrum. The method utilizes the effect of the reduced form of nicotinamide adenine dinucleotide on chromogens whose reduction produces a colored compound or, on the contrary, their coloration decreases. The various tetrazolium salts are most commonly used as chromogens, but less often the reduction of 2,6-dichlorophenolindophenol or the reduction of salts containing iron (III) or copper (II) is used.

This procedure cannot be used to determine creatine kinase activity. This enzyme is very quickly oxidized and loses its activity. Prior to its determination, it is necessary to reactivate by adding an excess of thiol compounds, for example glutathione, N-acetylcysteine or dithiothreitol. When measuring creatine kinase activity in ultraviolet light, thiol compounds do not interfere. However, when attempting to convert to the visible region of the above, the reducing effect of the thiol compounds on the chromogen appears, which thus non-specifically enhances the resulting coloration, which is not proportional to the creatine kinase activity.

The present invention provides a method for determining the catalytic activity of a thiol derivative-activated creatine kinase in the visible spectrum of the present invention in that excess thiol activators interfering with the reaction of the reduced form nicotinamide adenine dinucleotide with chromogen are removed by adding 2 to 4 moles of N-ethylmaleinimide. chromogen is then added and the resulting color is measured in the visible range of the spectrum.

The use of N-ethylmaleinimide blocks the undesired side reaction of thiol activators with chromogens by forming with them an inactive compound formed by the addition of their sulfhydryl group to the double bond of the heterocycle of N-ethylmaleinimide. to a reduced nicotinamide adenine dinocide, a solution of N-ethylmaleinimide is added.

Within 30 seconds, all of the free sulfhydryl groups are bound, which results in the inactivation of thiol activators and the setting of the creatine kinase-catalyzed enzyme reaction. In the next step, a color reaction can take place in which the chromogen is reduced only by a reduced form of nicotinamide adenine dinucleotide and the resulting color is proportional to the catalytic activity of creatine kinase. N-ethylmaleimide does not interfere in this color reaction. Measurements can be transferred to the visible range of the spectrum to extend the ability to perform activated creatine kinase in smaller laboratories not equipped with a spectrophotometer for ultraviolet light measurements.

Example 1

Using a Rosalki creatine kinase activity reagent containing 8 mmol / l glutathione, excess glutathione is inactivated by adding 0.4 mol / l N-ethylmaleinimide to a final concentration of 20 mmol / l. After 1 minute, the chromogen is added; its final test concentration is 0.8 mmol / l tetrazolium chloride and 0.32 mmol / l N-methylphenazinium methyl sulfate. The resulting color is measured at 500 nm.

Example 2

Using the Szasz creatine kinase activity reagent containing 20 mmol / l of N-acetylcysteine, the excess activator is inactivated by adding 0.4 mol / l of N-ethylmaleinimide to a final concentration of 40 mmol / l. After 1 minute, the chromogen is added to a final concentration of 1.0 mmol / l 3,3'-dianisole-4,4'-bis [2- (4-nitro-phenyl) -5-phenyltetrazolium chloride] and 0.4 mmol / l. 1 N-methylphenazinium methyl sulfate. The resulting color is measured at a wavelength of 530 nm.

Example 3

After serum electrophoresis on cellulose acetate and after incubation in the Szasz creatine kinase reagent, the film is transferred to a mmol / l N-ethylmaleinimide solution and after a minute a coloring solution containing

1.0 mmol / l of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide and 0.18 mmol / l of N-methylphenazinium methyl sulfate. The procedure serves to determine the activity of creatine kinase isoenzymes.

Claims (1)

SUBJECT Method for determining the catalytic activity of thiol derivative-activated creatine kinase in the visible spectrum, characterized in that excess thiol activators interfering in the reaction of the reduced form of nicotinamide adenine dinucleotide with chromogen are removed by adding 2 to 4 moles of N-ethylmaleinimide per mole of thiol activator. the chromogen and the resulting color are measured in the visible range of the spectrum.