JPH0553477B2 - - Google Patents

Description

Detailed Description of the Invention Field of Application of the Invention The present invention relates to reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as "NAD(P)").
More specifically, it is a method for quantifying NAD(P)H, which is characterized by reacting a sample containing NAD(P)H with resazurin in the presence of a catalyst and measuring the resulting change in absorbance in the visible region. ) Concerning a method for quantifying H.

Conventional techniques Conventional methods for quantifying NAD(P)H include (1) measuring its own ultraviolet absorption or fluorescence, (2) measuring NAD(P)H and a tetrazolium compound in the presence of diaphorase, etc. (3) Measuring the fluorescence of resorufin produced by the reaction of NAD(P)H and resazurin in the presence of a catalyst. [Meth. Enzymol. Vol. 41, pp. 53-56,
1975] is known.

The above method for measuring absorption in the ultraviolet region has the drawbacks that when using biological body fluids such as serum as a sample, it is interfered with by coexisting substances that absorb in the ultraviolet region and has low sensitivity. Although this method has high measurement sensitivity, it is not frequently used because fluorophotometers are expensive and not widely available. Furthermore, the above-mentioned method for measuring formazan dyes has disadvantages in that the dyes are poorly soluble and therefore adhere to the cell or tube of the photometer and that the measurement sensitivity is low.

NAD(P)H is a coenzyme for dehydrogenases and reductases, and is used in enzyme reactions when measuring these enzymes present in biological body fluids or when quantifying components in biological body fluids that serve as substrates for these enzymes. arose due to
Changes in NAD(P)H are measured. Currently, methods using oxidases are mainly used to quantify components in biological body fluids, but this method has the disadvantage of being easily interfered with by reducing substances such as ascorbic acid and bilirubin that coexist in the sample. be. Although the method using dehydrogenase or reductase is an advantageous method that is less susceptible to the effects of such reducing substances, it is currently not widely used for the reasons mentioned above.

Problems to be Solved by the Invention It is an object of the present invention to provide a simple and highly sensitive quantitative method that improves the drawbacks in the conventional quantitative determination of NAD(P)H.

Means for Solving the Problems As a result of intensive research into the quantitative method of NAD(P)H, the present inventors have found that there is a means to utilize a practically advantageous coloring dye. In the reaction to produce resorufin from resazurin and resazurin, we discovered a method for quantifying NAD(P)H by utilizing the property that resazurin and resorufin have an absorption maximum at a specific wavelength in the visible region. In other words, resazurin has a wavelength of 600nm on the slightly alkaline side.
Since resorufin is a blue substance with an absorption maximum near 570 nm and a red substance with an absorption maximum near 570 nm, NAD(P) can be determined by measuring the absorbance at wavelengths near the respective absorption maximums.
H is quantified. The reaction formula between NAD(P)H and resazurin is shown below.

Resazurin and sasorufin each have small extinction coefficients, so it may be difficult to quantify trace amounts using a commonly used spectrophotometer. In such cases, there is no need to dilute the reaction solution and a small volume (30 to 200μ) can be used.
) It is preferable to use a microplate type spectrophotometer that can perform measurements.

In the present invention, diaphorase and phenazine methyl sulfate are used as catalysts for the reaction of producing resorufin from NAD(P)H and resazurin, and diaphorase is particularly preferably used.

The quantification of NAD(P)H according to the present invention is performed using NAD(P)H.
It can be used to measure the activity of an oxidoreductase that uses (P)H as a coenzyme or to measure a substrate using this enzyme. Examples of such oxidoreductases include lactate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, α-glycerophosphate dehydrogenase, Examples include glycerol dehydrogenase, cholesterol dehydrogenase, glucose dehydrogenase, and pyrroline-5-carboxylic acid reductase.

Example 1 Quantification of NADH 10μ of 3mM resazurin, 10μ of 20U/ml diaphorase (manufactured by Boehringer), 5μ of 1M Tris-HCl buffer (PH8.0) and 0 to 0 as standard samples.
Mix 10μ of NADH at each concentration of 1.4mM, add water to make a total volume of 70μ, and then add 10μ of NADH at 37°C.
Incubation was carried out for 1 minute. Next, the absorbance of the reaction solution at 610 nm and 550 nm was measured using a microplate type dual wavelength spectrophotometer (manufactured by Corona Electric Co., Ltd., model MTP-12). The results are shown in the calibration curve in FIG. The same operation as above is performed using a sample of unknown concentration in place of the standard sample, and NADH in the sample is quantified by comparing the obtained absorbance with the calibration curve.

Example 2 Quantification of NADPH In Example 1, NADPH was used instead of NADH.
When the same procedure was performed using , the same calibration curve as shown in FIG. 1 was obtained. Similarly, by performing the same operation as above using a sample of unknown concentration and comparing the obtained absorbance with the above calibration curve, the
NADPH is quantified.

Example 3 Use for quantitative determination of ornithine 0.1U/ml ornithine-ketoacid aminotransferase (manufactured by Amano Pharmaceutical Co., Ltd.) 5μ, 0.1U/ml pyrroline-5-carboxylic acid reductase (manufactured by Amano Pharmaceutical Co., Ltd.) 5μ, 50mM α -ketoglutaric acid 5μ,
100μM dithiothreitol 5μ, 400μM
NADH 10μ, 1M Tris-HCl buffer (PH8.0) 5μ
And as a standard sample, 10μ of each concentration of ornithine from 0 to 0.3mM was placed in a microplate (manufactured by KUTSUKU Co., Ltd., disposable U type, 96 wells), water was added to make a total volume of 50μ, and the ink was incubated at 37°C for 1 hour. Basion. Then add it to the reaction solution.
After adding 10μ of 2mM resazurin and 10μ of 20U/ml diaphorase and incubating for an additional 5 minutes, the absorbance of the reaction solution at 610nm was measured using a microplate dual-wavelength spectrophotometer. The blank test was performed in the same manner except that pyrroline-5-carboxylic acid reductase was removed from the reaction solution. The measurement results are shown in the calibration curve in Figure 2. The same operation as above is performed using a sample of unknown concentration in place of the standard sample, and ornithine in the sample is quantified by comparing the obtained absorbance with the calibration curve.

Effects of the Invention The method of the present invention has made it possible to quantify NAD(P)H with high sensitivity by a simple method using a visible absorption photometer. Furthermore, as a ripple effect of the method of the present invention, a method for measuring oxidoreductases that is less susceptible to interference by reducing substances coexisting in a sample or a method for quantifying biological fluid components using the enzymes has been established.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the calibration curve for NADH quantification of the present invention, and FIG. 2 is a diagram showing the calibration curve for NADH determination of the present invention.
It is a figure showing the calibration curve in the quantitative determination of ornithine using the quantitative method of.

Claims (1)

[Claims] 1. Measurement of the change in absorbance in the visible region caused by reacting a sample containing reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate with resazurin in the presence of a catalyst. A characterized method for quantifying reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate. 2. The quantitative method according to claim 1, which measures the change in absorbance at 610 nm derived from resazurin. 3 Derived from resorufin produced by reaction
The quantitative method according to claim 1, which measures a change in absorbance at 550 nm. 4. The quantitative method according to claim 1, wherein the catalyst is diaphorase. 5. The quantitative method according to claim 1, wherein absorbance is measured using a microplate type spectrophotometer.