JPH01296999A - Measurement of living body specimen - Google Patents

Abstract

PURPOSE:To highly sensitively measure a dehydrogenase and a substrate thereof in a living body specimen for a clinical examination, etc., by treating NADH, etc., produced by the dehydrogenation reaction of the living body specimen with a pyrroloquinolinequinone, a peroxidase and luminol and subsequently measuring the luminous strength of the product. CONSTITUTION:When the respective contents of a dehydrogenase and a substrate thereof in a living body specimen are measured, reduced nicotinamide-adenine- denucleotide (NADH) or reduced nicotinamide-adenine-denucleotide phosphoric acid (NADPH) is treated with a pyrroloquinolinequinone, a peroxidase and luminol and the strength of the generated chemical luminescence is measured, thereby, quantifying the content of the dehydrogenase or the content of the substrate thereof in the living body specimen.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for measuring a biological sample, and more particularly, to a method for measuring a dehydrogenase content or a dehydrogenase substrate content in a biological sample. Involved.

[Prior art, problem to be solved by the invention] The dehydrogenase content and the dehydrogenase substrate content in a biological sample are determined to reduce the reduction produced by the dehydrogenase reaction by reducing ecotinamide-adenine-dihydrogenase. Nucleotide (hereinafter referred to as NAD)
It can be determined by measuring the amount of NADPH (denoted as H) or the amount of reduced nicotinamide-adenine dinucleotide phosphate (hereinafter referred to as NADPH and ligand).

Conventionally, the method for measuring NADH or NADPH is: ■ measuring NADH or NADH with absorbance at 340 nm
Method for measuring DPH ■For example, fuo-nadine metasulfate (PMS) and 1-methoxy-PMS
A method is known in which an electron carrier and formazan, such as E.g.

However, in these methods, NADH or N
The detection sensitivity of ADPH is low and insufficient for practical use, and there has been a need to develop a method that can measure with higher sensitivity.

[Means and actions for resolving intercolor]

The present inventors investigated various methods that could reproducibly and highly sensitively measure the content of dehydrogenase and dehydrogenase substrate in biological samples, and found that NADH produced by the dehydrogenation reaction Alternatively, by reacting pyrroloquinol/quinones, peroxidase, and luminol with NADPH, luminol is oxidized and chemiluminescence is generated, the intensity of which is 1 of NADH or 1 of NADP.
It has been found that the amount of NADH or NADPH is proportional to the amount of H, and that even a trace amount of NADH or NADPH can be measured. The present invention has been achieved based on this new knowledge.

That is, the present invention provides a method for measuring the content of dehydrogenase and dehydrogenase substrate in a biological sample:
NAD H or Ij N produced by dehydrogenation reaction
This is a biological sample measurement method in which biproquinoline is applied to ADPH and the intensity of chemiluminescence generated is measured.

Biological samples used in the present invention include samples derived from animals, plants, microorganisms, etc. Typical examples include blood, phlegm, and sputum, as well as extracts of various living organisms, and culture fluids of microorganisms and cells. and so on.

By applying the present invention to the measurement of various dehydrogenases contained in these samples, and to the measurement of substances that are substrates for dehydrogenases among the substances contained in these samples, these It becomes possible to measure substances with high sensitivity.

The process of measuring the respective contents of dehydrogenase and +/-hydrogenase substrates in a biological sample can be divided into the following two steps.

The first step is to extract NADH or NADP from the biological sample.
H (hereinafter referred to as NADH or NADPH collectively)
DCP)H may be written. This process also applies to NAD (P).

When measuring the base of dehydrogenase (dehydrogenase), add the substrate of this enzyme and NAD (P) to the reaction solution, and when measuring the amount of the substrate of dehydrogenase, add N.A.D.
By adding (P) to the reaction solution and reacting, N
ADH again t'! , NAD or 0 PH-firewood is generated.

Although this is a process that occurs, it is assumed that the reaction is based on a quadratic equation.

PQQ + NAD (P) H-4-02+ H+--)NAD
Q') + H2O2 It is also possible to perform the reaction in the first step and the reaction in the second step in the same reaction solution,
Depending on the biological sample, impurities mixed into the sample may be the main cause of light emission in the second step, so remove the mixed impurities before or after the first step. It is preferable to subject the sample obtained by the second step to a second step.

Treatment methods in this case include inactivation or removal of proteins such as enzymes, and treatment to remove metals such as iron, such as heat treatment, resin adsorption treatment, solvent denaturation, etc.
There is.

In the second step, as a method for measuring the generated chemiluminescence, a method using a general spectrometer for measuring radioactivity or the like is adopted.

In this way, the dehydrogenase content or dehydrogenase substrate content in biological samples can be reduced by 10% compared to conventional methods.
Measurements can be made with up to 100 times the sensitivity.

Furthermore, in this second step, 6-hydroxybenzothiazole, 2-cyano-6-hydroxybenzothiazole, or 2-(6-hydroxy-2-benzothiazolyl)thia-4-carboxylic acid ( By adding dehydrogenase (dehydrluciferin) or the like, it becomes possible to measure the dehydrogenase content or the dehydrogenase substrate content in a biological sample with about 10 times more sensitivity.

The concentration of the sensitizer is not particularly limited and is appropriately set depending on the reaction conditions and measurement time.

Examples of dehydrogenases include glucose dehydrogenase, fructose dehydrogenase, alcohol dehydrogenase, glucophosphate dehydrogenase, aldehyde dehydrogenase,
These include phosphogluconate dehydrogenase, malate dehydrogenase, incitrate dehydrogenase, glutamate dehydrogenase, and amine dehydrogenase.

In addition, examples of dehydrogenase substrates include 6-phosphoglufunic acid, malic acid, isocitric acid, glutamic acid, and amines.

The beer-quinoline quinones used in the present invention are pyrroloquinolisoquinone (also known as 2,7゜9-tricarboxy-IH-pyrrolo(2,3f)quinolinequinone-4,5-
PQQ salts, such as sodium salt of PQQ and potassium salt of PQQ, and derivatives of PQQ. Representative examples of PQQ derivatives include 1-methyl-PQQ, 7-zocarboxy PQQ
, 2-methyl ester-PQQ, and the like. These PQ
The amount of Class Q added is appropriately set depending on the type of sample, reaction conditions, and measurement time. PQQs are required in an equimolar amount to NAD (P), but in practical terms this amount is 10 to 200
Twice the amount is used. Note that there is no hindrance to using more than this.

Additionally, the peroxidase used is H2O2+AH
Any enzyme may be used as long as it has the activity of catalyzing 2→2HzO+A, and may be derived from animals, plants, or microorganisms. The concentration of these enzymes is not particularly limited, but ranges from 1 to 30 tJnit/-, especially from 1 to 30 tJnit/-.
15 units/al! is preferred. In addition, this 1Un
It was prepared by converting Img of purp gallin (2,3,4,6-tetrahydroxy-
5H-benzocyclohebuten-5-one) and 1. is shown.

Furthermore, the concentration of luminol (5-amino-2,3-dihydro R-1,4-phthalazinedione) used is not particularly limited, but is in the range of 1 to 30 μmol I e/1, more particularly 2 to 10 μmol I e/l is preferred. The pH of the reaction solution is 1! It is usually 4 to 10, and %5--e to 8→ is particularly preferable. There is no particular restriction on the reaction temperature, but it is usually 20
It is carried out in the range of ~40°C.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Using (A) described below as a reaction solution, pre-incubation was carried out at 25°C with stirring. Lum1n made by about 5 companies
The luminescence intensity of the reaction product liquid was measured using Escence Reader BLR, -102.

Total reaction solution 1-NAD The relationship between the (P)H concentration and the luminescence intensity is shown in FIG.

The NAD(P)H concentration in the reaction solution was approximately 5 μno
Quantification is possible up to I e/l.

Example 2 The following (B) was used as a reaction solution, and the mixture was incubated at 25° C. with stirring. Add 20 μl of each to a concentration of about 5 and react.
The luminescence intensity of the reaction product solution was measured using Ader BLR-102.

Reaction solution NAD (P)H aqueous solution at various concentrations
20μ! The relationship between total 1-NAD(P)H concentration and luminescence intensity is shown in FIG. 2.

The NAD(P)H concentration in the reaction solution is approximately 0.5 μm.
Quantification is possible down to ole/l.

〔Effect of the invention〕

It can be used for various clinical tests.

[Brief explanation of the drawing]

Both FIGS. 1 and 2 are graphs showing the relationship between NAD (P)H concentration and luminescence intensity. Patent applicant Kazuyoshi Nagano, representative of Mitsubishi Gas Chemical Co., Ltd.

Claims (1)

[Claims] When measuring the content of dehydrogenase and dehydrogenase substrate in a biological sample, pyrroloquinone is added to reduced ecotinamide-adenine-dinucleotide or reduced nicotisoamide-adenine-dinucleotide phosphate produced by the dehydrogenation reaction. A living body characterized by knowing the content of dehydrogenase or the content of a substrate for dehydrogenase in a biological sample by treating lysoquinones, peroxidase, and luminol and measuring the intensity of chemiluminescence generated. Sample measurement method.