CS240043B1 - Electron acceptor for determination of lactate concentration by cytochrome b £ - Google Patents

Abstract

The use of tetrazolium salt as an electron acceptor in the determination of lactate concentration using cytochrome b2 results in a significant increase in the sensitivity of the reaction, achieving a positive change in absorbance and, last but not least, the possibility of measuring the resulting color in the visible region of the spectrum. The use of the solution is particularly suitable for clinical biochemistry laboratories and other workplaces where lactate concentration is determined when assessing the severity of lactic acidosis in patients in serious condition, when determining the anaerobic threshold in patients after myocardial infarction and in athletes.

Description

The invention relates to the use of tetrazolium salt as an electron aceeptor in the determination of lactate concentration by cytochrome b ₂ .

Lactic acid resp. Lactate is an important metabolite produced as the end product of anaerobic glycolysis. Determination of lactate concentration in plasma is essential in patients in severe condition, especially in shock, where it helps to determine not only the diagnosis of lactic acidosis, but it is also used as an indicator of treatment success and disease prognosis. In patients after myocardial infarction, blood lactate concentration is used to determine the amount of exercise that the patient is still able to cope with by aerobic oxigenation, ie to determine the appropriate load for rehabilitation exercise. In athletes, the determination of the lactate concentration in the blood after physical exercise allows to assess the degree of training.

Usually one of two specific enzyme methods is used to determine the plasma lactate concentration. The first is the oxidation of lactate to pyruvate by nicotinamide adenine dinucleotide under catalysis by animal lactose dehydrogenase. The formation of a reduced form of nicotinamide adenine dinucleotide results in an increase in absorbance in the ultraviolet region of the spectrum. Since the reaction equilibrium is unfavorable for said reaction direction, one of the products, usually pyruvate, must be pumped off. For example, a transamination reaction with glutamate under alanine aminotransferase catalysis can be used. This principle is used in the work of Noll (in: Bergmeyer, HU, ed .: Methoden der Enzymatischen Analyze, 2nd Ed., Volume 2, Verlag Academy, Berlin 1970, pp. 1433 and 1437) and is also based on the determination of lactate concentration using the Lactat vollenzymatisch kit from Boehringer Mannheim. The need for the presence of two enzymes, coenzyme and another auxiliary substrate, makes the assay more expensive. Another disadvantage is the necessity of using a photometer with the possibility of measuring in the ultraviolet range of the spectrum; this photometer is

- 2 240 043 expensive and unavailable in smaller clinical biochemistry laboratories ·

The second method uses cytochrome b ₂ , usually isolated from aerobic yeast, such as baker's yeast. This enzyme also acts as lactate dehydrogenase and catalyzes the conversion of lactate to pyruvate. The enzyme does not need any coenzyme, and potassium fericyanide is used as an artificial electron acceptor. This method was first described by Wieland (Biochem. Z. 329, 1958, No. 7, p. 568-576) and was later used by other authors such as Durliat et al. (Clin. Chem. 22, 1976, p. 11, p. 1802-1805). The course of the reaction is observed as a decrease in absorbance in the short region of visible light due to the conversion of yellow ferriyanide to colorless ferrocyanide. The disadvantage of the assay is its low sensitivity and difficult automation with negative absorbance changes.

The principle of the electron acceptor in the determination of the lactate concentration by cytochrome b ₂ is the use of the tetrazolium salt. This compound, commonly used as a redox indicator to detect a reduced form of nicotinamide adenine dinucleotide or sulfhydryl groups, is in this case reduced with lactate under the catalytic action of cytochrome b ₂ to formazan; the increase in absorbance is measured in the visible region of the spectrum.

The advantage of this assay is its simplicity and high sensitivity, almost ten times higher than when using ferriic anhydride. As the absorbance increases, the results can be easily evaluated automatically. The resulting color is measured in the visible region of the spectrum, making the assay available to those laboratories that are not equipped with a spectrophotometer for ultraviolet light measurements. Isolation of cytochrome b ₂ is cheap and easy to implement for simplicity of procedure.

Example 1

When determining the lactate concentration using cytochrome b ₂ ee, the electron acceptor used is 3,3'-dianisole-4,4'-bis [2- (4-nitrophenyl) -5-phenyltetrazolium chloride] at a final concentration of 0.4 mol / l and as electron carrier N-methylphenazinium methyl sulfate at a final concentration of 0.15 mmol / l. The resulting color is measured at a wavelength of 530 nm.

- 3 Example 2 ²⁴⁰ «43

Gradient polyacrylamide gel electrophoresis is followed by incubation in a solution containing lactate at a concentration of 50 mmol / l, 3- (4-, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide at a concentration of 2.0 mmol / l and N- methylphenazinium methyl sulfate at a concentration of 0.2 mmol / l. The procedure serves to characterize lactate dehydrogenase after its isolation from yeast.

Claims (1)

SUBJECT OF THE INVENTION 240 043 Use of tetrazolium salt as an electron acceptor in the determination of lactate concentration using cytochrome bg