SU1635139A1 - Method for determination of water-soluble melanin - Google Patents

Abstract

This invention relates to methods for biochemical analysis, in particular to methods for determining the concentration of melanin pigments. The purpose of the invention is to improve the accuracy of the method. The amount of melanin is determined by the degree of inhibition of the nitro tetrazole reduction reaction in formaean in a weakly alkaline medium, pH 8.2 in the presence of phenazine methosulfate and nicotinamide adenine dinucleotide (NADHa), or by accelerating the adrenaline autooxidation reaction in an alkaline medium pH 10.2. The amount of melanin is determined spectrophotometrically by the change in the initial rate of formation of the reaction product as compared to the control. (In the first case, by slowing down the initial rate of formazan formation at 560 nm in the second, by accelerating the initial rate of adrenochrome formation at 500 nm). 3 tab.

Description

The invention relates to methods of biochemical analysis, in particular to methods for determining the concentration of natural and synthetic melanin pigments, and can be used in research and medicine.

The purpose of the invention is to improve the accuracy of the method.

The method is carried out as follows.

Spectrophotometric methods to detect the superoxide radical in an aqueous medium are used to quantify the water-soluble melanin. The amount of melanin is determined by the degree of inhibition of the nitro-blue tetrazole reduction reaction (NST) in formazan in a weakly alkaline medium, pH 8.2 in the presence of phenaine methyl sulphate and reduced

nicotinamide adenine dinucleotide - NADH2 or by accelerating the reaction of adrenaline autooxidation in an alkaline medium pH 10.2. The amount of melanin is determined spectrophotometrically by the change in the initial rate of formation of the reaction product as compared to the control. In the first case, by braking the initial formation rate of formaean (VH) at 560 nm

(VH. T 1 min), in the second - by

accelerating the initial rate of adrenochrome (VH) formation at 500 nm

(, t 1 min). Concentration

melanin is calculated from calibration curves.

PRI me R 1. Determination of the concentration of melanin using the nitro-blue tetrazole reduction method. AT

is smiling

Oj

ate

with y

a 10 mm thick spectrophotometric cuvette containing 50 μM nitro-blue tetrazole (0.5 ml), 4.5 μM phenazine metosulfate (0.2 ml) in 0.05 M pyrophosphate buffer, pH 8.2 with 0.69 μM EDTA. 0.2 ml of an aqueous solution containing melanin pigment is added. Immediately before spectrophotometry, 384 µM (1 ml) of reduced nicotinamide adenine dinucleotide - NADH2 was added to the reaction mixture. The total volume of the reaction mixture is 3 ml. The control is a sample that does not contain melanin. All solutions, except the buffer, are prepared on the day of analysis and are kept in a dark dish.

The results of determination of melanin pigments obtained from grape marc and from silkworm excreta are given in Table. one.

Example 2. Determination of melanin concentration using the method of autooxidation of adrenaline in an alkaline medium. To the spectrophotometric cuvette with a thickness of 10 mm, containing 2.3 ml of a 0.2 M solution of Na-carbonate buffer pH 10.2, 0.2 samples were added and immediately before spectrophotometry with 0.5 ml of a 5.4 x 10 solution of adrenaline (hydrotartrate) at 0, 2% citric acid. The amount of melanin is determined by the value of the initial rate of formation of the product of the oxidation of adrenaline — adrenochrome at 500 nm or

by the percentage of reaction acceleration with respect to the control one, using calibration curves.

The results of the determination of melanin pigments from grape marc and from the excretions of the silkworm larvae are given in Table. 2

In tab. 3 presents data confirming a higher accuracy of the proposed method in comparison with the known method.

Thus, the use of the proposed method provides increased accuracy compared with the method of the prototype.

Claims (1)

The method of determining water-soluble melanin, including sample processing and spectrophotometric recording, is due to the fact that, in order to improve the accuracy of the method, the sample is added to the phenazine methosulfate-reduced superoxide radical generation system and nicotinamide adenine dinucleotide in the presence of a nitro blue tetrazolium arm or in the presence of nitrous blue tetrasolium arm or in the presence of nitrous blue or glycerone-reduced and nucleic acid. and the melanin content is determined by the degree of inhibition of the rate of reduction of nitro blue tetrazolium or by the degree of increase in grow to form adrenochrome. Table 1 table 2 Table 3