JPS6191570A - Assay of reducing type nicotinamide adenine dinucleotide and reducing type nicotinamide adenine dinucleotide phosphate - Google Patents

Abstract

PURPOSE:To assay reducing type nicotinamide adenine dinucleotide phosphate 'NAD(P)H' handily with a high sensitivity, by making reducing type nicotinamide adenine dinucleotide or NAD(P)H react with resazurin to measure the absorbance at the wavelength near the maximization of absorption. CONSTITUTION:NAD(P)H is made to react with resarzurin using a catalyst such as diaphorase to from resorufine. The resazurin is a blue substance having the maximization of absorption near 600nm on the slight alkaline side while the resorufine is a red substance having the maximization of absorption near 570nm. Therefore, the assay of NAD(P)H can be done by measuring the absorbance at the wavelength near the respective maximization of absorption. In the measurement of the absorbance, for example, a microplate type 1-wavelength spectrometer is used.

Description

Detailed Description of the Invention Field of the Invention The present invention relates to a method for quantifying reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as rNAD (P)H). is NAD (P
)N, 6, characterized in that a sample containing H is reacted with resazurin and the resulting change in absorbance in the visible region is measured.
．． This invention relates to a method for quantifying D(P)H.

Prior Art Conventional methods for quantifying NAD (P)H include (1) measuring its own ultraviolet absorption or fluorescence;
2) A method in which NAD(P)H and a chromogen such as a tetrazolium compound are reacted in the presence of diaphorase to form a formazan dye, and the absorption in the visible region is measured; and (3)
A method for measuring the fluorescence of resorufin produced by the reaction of NAD(P)H and resazurin in the presence of a catalyst [Meth, Enzymology]
ymol, ) Vol. 41, pp. 53-56, 1975]
It has been known.

The above method for measuring absorption in the ultraviolet region has the drawbacks that when biological body fluids such as serum are used as samples, 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 used. In addition, the method for measuring the formazan dye described above has disadvantages in that the dye is poorly soluble and therefore adheres to the cell or tube of the photometric needle and that the measurement sensitivity is low.

NAD (P)H is a coenzyme of dehydrogenase and reductase, and it is used in the measurement of these enzymes present in biological body fluids or when quantifying components in biological body fluids that are substrates for these enzymes. Changes in NAD (P)H are measured. Currently, methods using oxidases are most commonly used to quantify components in biological body fluids, but this method has the disadvantage that it is easily interfered with by reducing substances such as ascorbic acid and bilirubin that coexist in the sample. There is. 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 of the conventional quantitative determination of NAD (P)H.

Means for Solving the Problems As a result of intensive research into the quantitative determination of NAD (P)H, the present inventors have found that there is a method that utilizes a practically advantageous coloring dye. In the reaction to produce resorufin from resazurin and resazurin, we have 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. That is, resazurin is a blue substance that has an absorption maximum around 600 nm on the slightly alkaline side, while resorufin is a red substance that has an absorption maximum around 57 Onm, so by measuring the absorbance at wavelengths near the respective absorption maximums, NAD (P)
H is quantified. The reaction formula between NAD (P)H and resazurin is shown below.

] - Because resazurin and resorufin each have a small extinction coefficient, it may be difficult to quantify a trace amount using a commonly used spectrophotometric needle. In such a case, it is preferable to use a microplate type spectrophotometer that can measure the step volume (30 to 20(1)) without requiring dilution of the reaction solution.

In the present invention, the catalyst used in the reaction to produce resorufin from NAD (P)H and resazurin is:
For example, diaphorase and phenazine methyl sulfate are used, and diaphorase is particularly preferably used.

The determination 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 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, glycerol dehydrogenase, cholesterol dehydrogenase, glucose dehydrogenase,
Examples include virolin-5-carboxylic acid reductase.

Example I Quantification of NADH 3mM resazurin 10pf! , 201J/ml! Diaphorase (manufactured by Boehringer) 10 g, IMI-Lis-HCl buffer (pH 8,0) 54 and 0 to 0 as a standard sample
NADHlom at each concentration of 1.4 mM was mixed, water was added to bring the total volume to 70 degrees, and the mixture was incubated at 37 degrees Celsius for 10 minutes. Next, a microplate type three-wavelength spectrophotometer (1MT P-
12 type) of the reaction solution at 610 nm and 550 nm.
The absorbance at m was measured. The results are shown in the calibration curve in FIG. Perform the same operation as above using a sample of unknown concentration instead of the standard sample, and compare the obtained absorbance with the calibration curve to obtain a sample.

NADH in it is quantified.

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

Example 3 Use for quantitative determination of ornithine 0, IU/me ornithine-ketoacid aminotransferase (manufactured by Ohno Pharmaceutical Co., Ltd.) 54.0-10/ml! Pyrroline-5-carboxylic acid reductase (manufactured by Ohno Pharmaceutical Co., Ltd.) 5pR,
50mM α-ketoglutarate 5g, 100μM dithiothreitol 5+, 400μM NADH10#, IM
) Lis-HCl buffer (pH 8,0) 5u and ornithine 1 (l
was placed in a microplate (manufactured by Tack, disposable U-type, 96 wells), and water was added to make the total volume 5M.
Incubation was for 1 hour at 37°C. Then, 2mM resazurin was added to the reaction solution at 10 degrees and 200/me.
After adding 10 μ of diaphorase and inking for another 5 minutes, the absorbance of the reaction solution at 610 nm was measured using a microplate type three-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 a calibration curve for NADH quantification according to the present invention, and FIG. 2 is a diagram representing a calibration curve for ornithine quantification using the NAD (P)H quantification method of the present invention.

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. 3 550 derived from resorufin produced by reaction
The quantitative method according to claim 1, which measures changes in absorbance in 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.