FR2520380A1 - VERY SENSITIVE METHOD FOR DETERMINING A SUBSTANCE BY CO-ENZYME RECYCLING - Google Patents

Abstract

A highly sensitive assay is described and is characterised by the following reaction stages: 1) a main reaction system in which the coenzyme is transformed according to the amount of the substance to be determined by the action of the oxidoreductase for the said substance in the presence of coenzyme NAD<+> or NADH, or NADP<+> or NADPH; 2) a decomposing reaction system in which the unreacted coenzyme is decomposed and neutralised at the end of the main reaction system; 3) a cycling reaction system in which the reaction is enhanced by means of coenzyme cycling, and which consists of a combination of the oxidoreduction reaction which transforms the excess substrate and coenzyme according to the amount of the substance to be determined, and of a converting reaction of tetrazolium salt to formazan in the presence of diaphorase or of an electron transfer substance and 4) a colorimetric measurement of the formazan formed in this way.

Description

The present invention relates to a very sensitive detection method. Heretofore, as very sensitive determination methods, the fluorescence method, the luminescence method and others have been known.

However, in these methods, samples such as body fluids cause errors in determination, such as setting value and extinction, due to impurities in the body fluid.

Methods of determination by recycling coenzyme are well known. However, in the prior method, an indicating reaction is required after the recycle reaction, which causes a complicated implementation, and the indicating reaction itself is influenced by the substance other than the substance. to be determined.

In addition, a special fat reaction vessel and extremely small reaction volume are required.
In addition, in the prior process, the unreacted coenzyme to be decomposed is decomposed by adding an acid or an alkali, heating and neutralizing the reaction mixture, and in these operations it is important to add the exact amount of acid or alkali in order to neutralize the reaction mixture and, if the pH of the neutralized reaction mixture changes, this affects the recycle rate. These phenomena causing errors in the determination.

It has been found that in the recycle reaction, the recycle reaction itself can be used as an indication reaction, which makes unnecessary the special use of an indication reaction and corresponding reagents. In addition, turn point determination and speed determination methods become possible and there is no longer a need for a special reaction vessel.

It has also been found that the adjustment of the pH is easily effected by adding an excess of the lower alkyl ester of formic acid so as to avoid that the recycle rate is influenced by changes in pH on neutralization.

An object of the present invention is to provide a method of determination in which the recycle reaction itself is the indicating reaction, without the need for taps and reagents for the indicating reaction, and without influence of substances other than the substance to be determined.

Another object of the present invention is to provide a determination method not only for a endpoint determination method, but also for a rate determination method. Another object of the present invention is to provide a determination method. simple, not requiring a special reaction vessel; - not requiring an extremely small reaction volume and capable of being carried out in a conventional constant temperature cabinet.

Yet another object of the present invention is to provide a method of determination in which the pu of the reaction mixture can be easily adjusted by adding an excess of lower alkyl ester of formic acid so as to avoid error. measurement resulting from the fact that the recycling rate is influenced by a variation in the pH in the event that the exact addition of the amount of acid or alkali is not carried out during the neutralization of the reaction mixture after decomposition of unreacted coenzyme.

Yet another object of the present invention is to provide a determination method which does not require operations of purification and concentration of the substance to be determined,
Embodiments of the principles of the present invention are as follows.

où AH2 ou A : substance à déterminer
NAD(P) : NAD+ ou NADP+
NAD(P)H : forme réduite de NAD+ ou de NADP+ (2) Réaction de décomposition

(1) Main reaction system

where AH2 or A: substance to be determined
NAD (P): NAD + or NADP +
NAD (P) H: reduced form of NAD + or NADP + (2) Decomposition reaction

où M : métal alcalin et R : groupe alcoyle inférieur.Neutralization
Neutralization of alkali;

where M: alkali metal and R: lower alkyl group.

(3) Cyclization reaction

In the present invention, the oxidoreductase used in the main reaction is not limited and has an action on the substance to be determined as a substrate at the same time as N D (P) or NAD (P) H as a coenzyme. These enzymes can be a new enzyme or a known enzyme. Examples of previously known enzymes and substrates are found in "Enzyme Handbook" (Shiro Akabori Ed.

Asakura publ. Co, Tokyo).

The amount of the reaction mixture varies between 10 ml and 3 ml. The amount of the coenzyme is 0.1 mole to 10 mmol. The minimum measurable amount of the substance to be determined is 10 8 - 10 15 moles and the amount of coenzyme is an amount in excess of the ratio of action on the substance to be determined.

The above reaction occurs at the optimum pH of the oxidoreductase and in an optimum temperature range. Then, the unreacted coenzyme is decomposed simultaneously with the completion of the main reaction. In this decomposition reaction system, in the case of an oxidized form of NAD or NADP + coenzyme, an alkali is added so that a reduced form of coenzyme remains,
NADH or NADPH, from the main reaction.

Also, we get to what remains NAD + or NADP + by adding an acid and heating, in the case where the unreacted coenzyme is NADH or NaDPH. Examples of alkalis are usually an alkali metal hydroxide such as aqueous sodium or potassium hydroxide. In addition, aqueous lithium hydroxide, sodium carbonate or potassium carbonate can be used.

The concentration of the alkali should be such that the pH of the solution is an alkaline pH above 12. A preferable concentration is between 0.001M and 0.5M.

The heating conditions vary depending on the alkali concentration, the heating temperature or the duration of the heating. NAD or NADP is completely decomposed after 10 minutes at 1000C in a 0.1N solution of
NaOH.

Examples of acids are such that the acidity of the aqueous solution corresponds to a pH below 2, as in the case of hydrochloric acid or sulfuric acid. A preferable concentration of the acid is between 0.00lu and 0.5M. The heating conditions vary depending on the concentration of the acid, the heating temperature and the duration of the heating. For example, NADH or
NADPH can be decomposed after 3 minutes at 500C in a 1N HCl solution.

In the neutralization reaction step after decomposition of the unreacted coenzyme, in the case where NAD + or NADP is decomposed by an alkali, the reaction mixture is decomposed by adding a lower alkyl ester formic acid. Examples of lower alkyl esters of formic acid are methyl formate, ethyl formate, propyl formate or butyl formate. In the conventional method, the pH of the reaction mixture is dispersed if the pH value of the reaction mixture is dispersed. the addition of acid is not carried out exactly, and the duty cycle varies to cause an error in the determination value,
However, in the above neutralization reaction, even if an excess of the lower alkyl ester of formic acid is added over the amount used in the neutralization, for example ten times the amount needed for the neutralization , only the amount of formic acid lower alkyl ester corresponding to the amount of alkali can react, and therefore exact neutralization of the reaction mixture can be carried out and the pH adjustment can be carried out easily.

In the case where NADH or NADPH is decomposed by an acid, a slight excess of alkali is added for neutralization, after which a lower alkyl ester of formic acid is added as indicated above for neutralization and then the mixture of reaction can be neutralized exactly.

An example of the alkali is usually an aqueous solution of an alkali metal hydroxide. The next step of the reaction in the present invention, namely the amplification reaction by recycling coenzyme, can be carried out by combining the oxidoreductase system, which acts on the remaining transformed coenzyme corresponding to the substance to be determined. and an excess of substrate, and the transformation system in which the tetrazolium salt is converted to formazan by the diaphorase or the electron transfer substance. The oxidoreductase of sisttr, the above is not limited as to its origin and is a dehydrogenase which requires a coenzyme NAD (P) + ct forms NAD (p) H by acting on an excess of specific substance.

Examples of these enzymes and substrates are found in the "Enzyme Handbook" mentioned above.

The amount of enzyme used depends on the activity of the enzyme, the type of substrate and the rate of recycling of the coenzyme.

The molar amount of substrate must be in large excess relative to the recycled coenzyme and its concentration may be greater than that corresponding to the maximum reaction rate of the oxidoreductase and is preferably 0.1-50 U / ml in the reaction mixture. An example of the electron transfer substance is a substance having oxidative activity on NAD (P) H to give NAD (P) and without detrimental action on the recycling reaction of the coenzyme, for example, methosulfate. phenazine, Meldola's Blue and pyrocyanine. Its concentration can be chosen according to the recycling rate and is preferably 1 µg to 1 mg per ml of the reaction mixture. Examples of tetrazolium salt 3.3 '- (3.3' dimethoxy-4,4 '-diphenylene) bis / 2 - (p-nitrophenyl) -5phenyl-tetra-zoliu ~ / (NTB) chloride, 2- (p-nitropbenyl) -3- (p-iodophenyl) -5-phenyltetrazolium ( INT) or 2- (4 ', 5'-dimethyl-2'-thiazolyl) -3,5-diphenyltetrazolium bromide (4,5-MTT). The concentration of the tetrazolium salt is somewhat limited by the solubilities of the tetrazolium salt and the formazan finally formed and is 3 to 100 mg per ml of the reactant.

The recycling reaction can be carried out between room temperature and 37 ° C., preferably at 30-370 ° C. The reaction can be stopped at an estimated time by adding an acid such as hydrochloric acid or phosphoric acid. Preferably, a surfactant can be added so as to avoid precipitation of formazan.

An example of a surfactant is a nonionic surfactant such as Triton X-100 or Adekarol S0-14 (trade names). The concentration of the surfactant is 0.01-3% based on the reactant.

Addition of the surfactant provides increased sensitivity of the determination and stabilization of the formazan pigment. The colorimetric determination of the formazan pigment thus formed can be carried out by measuring the optical density (OD) at the specific absorption wavelength of the formazan, for example 500-550 nm.

A substance can be determined by the method explained above. According to the method of the present invention, not only the turn point determination, but also speed determination can be performed. The following examples illustrate the present invention, but should not be construed as limiting it.

Example 1
Quantitative determination of NAD Reagents (1) 0.2M phosphate buffer solution (pH 8.0) (2) INT-Adekatol / INT solution (100 mg) dissolved in a
2% Adekatol-SO-145 solution (100 ml) 7 (3) Diaphorase or electron transfer substance.

1) 100 U / ml diaphoraz (NADH) solution.

2) solution of phenazine methosulfate (PMS) 100 mg /
20 ml.

3) 10 mg / 20 ml pyrrocyanine solution.

4). Meldola Blue solution 10 mg / 20 ml, (4) Ethanol (5) Alcohol dehydrogenase 250 U / ml, (6) 10 mM solution of NAD (In the main reaction7
NAD is formed and unreacted NADH is decomposed
laid by an acid and by heating, then neutralized
by an alkali, and then completely neutralized with
ethyl formate medium).

(7) 0.1 N HCl
Operating mode
Into each of a number of separate test tubes are placed phosphate buffer (0.5 ml), INT
Adekatol (0.2 ml), methanol (20 ssl), water (30 ssl) and alcohol dehydrogenase (0.1 ml), respectively.

Diaphorase (NADH) or electron transfer substance (50 µl) is added thereto and incubated at 37 ° C for 3 minutes. Then a 10 zM solution of
NAD (0, 10, 20, 40, 60, 80 or 100 µl), respectively, each volume is adjusted to a total of 1.0 ml and incubated at 37 ° C for exactly 10 minutes. 0.1 is added. N HCl (2 ml) in each tube to stop the reaction and the OD is measured at 50 nm Water (0.1 ml) is added in place of NAD as a control, Result
The result is shown in Fig. I, where the amount of NAD together with the diaphorase or the electron transfer substance and the absorption optical density have good linearity. The result shows that the recycling reaction is exact and is very sensitive.

Example 2
Effect of the oxidoreductase used in the recycling reaction
Reagents (l) 0.2 M phosphate buffer pH 8.0 (2) same INT-Adekatol solution as in example l (3) diaphorase solution (NADH) (4) substrate solution
1) 100 mM ethyl alcohol
2) 100 mM glycerol-3-phosphate solution
3) 100 mM lactic acid
4) 100 mM malic acid (5) oxidoreductase
1) solution of dehydrogenated alcohol (ADH) 25000 U / ml
2) glycerol-3-phospbate dehydrogenase solution
(G-3-PDH) 600 U / ml
3) solution of lactate dehydrogenase (LDH) 1500 U / ml
4) solution of acetate dehydrogenase (MDH) 4600 U / ml
5) 10 zM solution of NAD +
6) 0.1 N HCl
Operating mode
Into each of a number of separate test tubes are added phosphate buffer (0.5 ml),
INT-Adekatol (0.2 ml), diaphorase (NADH) (50 l), substrate solution (50 l), oxidoreductase acting on the above substrate (5 tii) and water ( 95 tii), separately, and incubated at 37 ° C for 3 minutes.

Add 10 sM solution of NAD + (0, 10, 20, 40, 60, 80 or 100 til) to each of the enzymes, respectively, adjust to a total volume of 10 ml and incubate at 37 C for exactly 10 minutes. 0.1 N HCl (2 ml) was added to each tube to stop the reaction and the OD was measured at 500 nm. Water (100 tiI) was used in place of the NAD + solution as a control.

Result
The result is shown in Figure 2. In each oxidoreductase, good linearity is shown and indicates the exact recycling reaction and high sensitivity.

Example 3
Quantitative determination of NADP Reagents (1) 0.2 M phosphate buffer pH 8.0 (2) INT-Adekatol solution [INT (100 mg) dissolved in a
2% Adekatol S0-145 solution (100 ml) 1 (3) NADPH-diaphorase (NADpH) solution 100 U / ml (4) substrate solution
1) 100 mM glucose-6-phosphate (G-6-P)
2) 100 mM glutamic acid (GA) (5) oxidoreductase solution
l) glucose-6-phosphate dehydrogenase solution
(G-6-PHD) 1000 U / ml
2) glutamate dehydrogenase (GlDH) solution
1000 U / ml (6) 10 sM solution of NADP + (7) 0.1 N HCl
Operating mode
In separate test tubes are added phosphate buffer (0.5 ml), INT-Adekatol (0.1 ml), diaphorase (NADPH) (100 til), substrate (50 µl), enzyme (50 l) and water (30 l), respectively, and incubated at 37 C for 3 minutes, followed by addition of -10 ssM solution of NADP + (0, 10, 20, 40, 60, 80 and 100 µl, respectively. The total volume is adjusted to 1.0 ml by adding water and incubated at exactly 37 ° C. for 10 minutes. 0.1 N HCl (2 ml) is added to stop reaction and the OD was measured at 500 nm Water (100 µl) was added in place of the NADP solution as a control.

Result
The result is shown in Fig. 3, where good linearity is obtained, and NADP + can be determined with high sensitivity together with glucose-6phosphate dehydrogenase and glutamate dehydrogenase.

Example 4
Quantitative determination of malic acid
Sample
Malic acid solution (malic acid: 0, 1, 2, 4, 6, 8
and 10 ssM) 20 l Reagents (1) Reagent I: mixed solution of 0.2 M phosphate buffer
pH 8.0 (0.2 mL), 10 mM NAD + solution
(0.1 ml) and ddshydro- malate solution
genase (120 U / ml) (100 pl) (2) 0.2 N NaOH (3) ethyl formate (4) Reagent II: mixed solution of 0.5 M phosphate buffer
pH 8.0 (0.1 mi), ethyl alcohol (10 l),
alcohol dehydrogenase (250 / ml) (50 tii),
diaphorase (NADH) (100 / mi) (100 tii),
lz solution of NTB (20 l) and solution
of Triton X-100 -20 tii) (5) 0.1 N HCl
Operating mode:
Samples are added to separate test tubes, Reagent I (0.2 ml) is added and then incubated at 37 C for 15 minutes. 0.2 N is added
NaOH (0.1 ml) to the reaction mixture, heated at 100 C for 10 minutes, cooled immediately, then neutralized with ethyl formate (20 ss After incubation at 370C for 5 minutes, reagent II is added (0.3 ml) and incubated at 37 ° C. for exactly 10 minutes 0.1 N HCl (2.5 ml) was added to stop the reaction and the OD was measured at 550 nm. water (100 l) in place of the NAD solution as a control.

Result
The result is shown in Fig. 4. The amount of malic acid is related to the OD with less error. Malic acid was determined with high sensitivity.

Example 5
Quantitative determination of gamma-aminobutylic acid
Sample:
Human serum (50 µl) in which gamma-aminobutylic acid (GABA) (0, 10, 20, 30, 40 and 50 µM) has been added -.

Reagents (l) Reagent I: mixed solution of 0.1 M pyrophos buffer
phate, pH 8.3 (1.0 mL), 10 mM solution of
NADP (0.2 mi), 0.1 M alpha solution
ketoglutarate (10 ml) and GABASE (2 U / ml,
0.4 ml, commercially available enzyme
derived from Pseudomonas fluorescens) (2) 0.5 N solution of NaOH (3) ethyl formate (4) Reagent II: mixed solution of INT-Adekatol solution
[0.1 ml, INT (100 mg) dissolved in a
2 z solution of Adekatol SO-145 (100 ml) 7,
diaphorase solution (NADPH) (100 U / ml,
1.0 ml), 0.1 M-glucose-6-phos solution
phate (0.2 ml) and glucose-6 solution
phosphate dehydrogenase (100 U / ml, 2.0 tii).

Operating mode
Reagent I (0.35 ml) is added to the sample and incubated at 37 ° C for 30 minutes. 0.5 N is added
NaOH (0.1 ml) and heated at 1000C for 10 minutes.

After cooling, ethyl formate (20 L) is added for neutralization and incubated at 37 ° C for 5 minutes. Reagent II (0.2 ml) is added and incubated at 37 ° C for exactly 10 minutes. 0.1 N HCl (2 ml) was added to stop the reaction and the OD was measured at 500 nm. A control without GABA is used.

Result
The result is shown in Fig. 5. The amount of GABA in human serum can be spotted on the line representing the linear relationship with the OD value. GABA can be determined with high sensitivity and without protein separation.

Claims (18)

1. Method for the very sensitive determination of a substance, characterized in that it comprises the following reaction steps 1) main reaction system transforming the coenzyme corresponding to a quantity of the substance to be determined by the oxidoreductase actioh for this substance in the presence of coenzyme NAD or NADH or NADP or NADPH 2) decomposition reaction system breaking down and neutralizing unreacted coenzyme at the end of the main reaction system 3) recycling reaction system amplifying the reaction by means of co-enzyme recycling consisting of a combination of an oxidation-reduction reaction which acts on excess substrate and the transformed co-enzyme corresponding to the amount of substance in determining and a reaction of transformation of tetrazolium salt into formazan in the presence of diophorase or an electron transfer substance; and 4) colorimetric measurement of the formazan thus formed. 2. Determination method according to claim 1, characterized in that the molar amount of coenzyme in the main reaction system is in excess with respect to the molar amount of substance to be determined. 3. Determination method according to claim 1, characterized in that the unreacted coenzyme in the case of NAD and NADP is decomposed by adding an aqueous alkali metal hydroxide and heating, and neutralization. 4. Determination method according to claim 3, characterized in that the concentration of the aqueous alkali metal hydroxide is a concentration giving a pH of more than 12. 5. Determination method according to claim 4, characterized in that the alkali metal hydroxide is sodium hydroxide or potassium hydroxide. 6. Determination method according to claim 3, characterized in that the heating is carried out until complete decomposition of NAD + or NADP +. 7. Determination method according to claim 3, characterized in that the neutralization is carried out by adding a lower alkyl ester of formic acid. 8. Determination method according to claim 1, characterized in that the unreacted coenzyme, in the case of NADH or NADPH, is decomposed by adding an aqueous acid and heating, and neutralization. 9. Determination method according to claim 8, characterized in that the concentration of the aqueous acid is a concentration giving an acidity corresponding to a pH below 2. 10. Determination method according to claim 9, characterized in that the acid is a substance giving an acidity below pH 2 in its aqueous solution. 11. Determination method according to claim 10, characterized in that the acid is hydrochloric acid or sulfuric acid. 12. Determination method according to claim 8, characterized in that the heating is carried out until complete decomposition of NADH or NADPH. 13. Determination method according to claim 8, characterized in that the neutralization is carried out by adding a small excess of alkali metal hydroxide solution and immediately adding a lower alkyl ester of formic acid. 14. Determination method according to claim 13, characterized in that the alkali metal hydroxide is sodium hydroxide or potassium hydroxide. 15. Determination method according to one of claims 7 and 13, characterized in that the lower alkyl ester of formic acid is methyl formate, ethyl formate, propyl formate or formate. butyl. 16. Determination method according to claim 1, characterized in that the electron transfer substance in the recycling reaction is phenazine methosulfate, Meldola blue or pyrocyanine. 17. Determination method according to claim 1, characterized in that a surfactant is added. 18. Determination method according to claim 17, characterized in that the surfactant is a nonionic surfactant.