JPH0141223B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for eliminating the influence of ascorbic acid when quantifying various components present in body fluids. Diagnosis of various diseases by quantifying various components in body fluids is currently widely practiced, and in recent years colorimetric assays using enzymatic reactions have been increasingly used as this measurement method. For example, (1) a method in which hydrogen peroxide is produced via an oxidase reaction system and is allowed to act on an oxidizable chromogen in the presence of peroxidase to colorimetrically quantify the produced pigment compound; (2) dehydration; NADH or
There is a method in which NADPH is generated, this is allowed to act on a tetrazolium salt in the presence of an electron transport system, and the formazan produced by reduction is measured colorimetrically. However, since ascorbic acid is present in body fluids, the strong reducing properties of ascorbic acid affect the oxidation and reduction systems in the colorimetric method, resulting in measurement errors.
Ascorbic acid exists in the serum of normal people at around 1.2 mg/dl, but as large amounts of ascorbic acid are used to treat diseases, the concentration in the serum has increased significantly, and the impact on measurements is extremely large. . In the quantitative method (1) above, the dye produced by the condensation of the oxidizable chromogen is reduced, giving a negative error to the measured value, and in the quantitative method (2), the tetrazolium salt is reduced. Generates formazan and gives a positive error to the measured value. In this way, the influence of ascorbic acid is much greater than that of glutathione and bilirubin present in serum, and the effect of ascorbic acid is much greater than that of glutathione and bilirubin present in serum. It is strongly desired to remove this influence during measurement. Conventionally, in this regard, a method using ascorbic acid oxidase (ASOD), a type of oxidizing enzyme, was generally used, but although the method using ASOD can reduce the effect of ascorbic acid, it is not possible to completely eliminate it. is difficult and also ASOD
Since it is an enzyme, it is very expensive, has poor stability, and is strongly affected by mixed substances. The present inventors have repeatedly investigated various measurement methods that use stable chemical substances that are highly effective in removing the effects of ascorbic acid in place of ASOD.
General formula (A) or (B) below (In the formula, R ₁ to ₅ are each H, an alkyl group, a carboxyalkyl group, or a hydroxyalkyl group, and X is an ethylene group; an ethylene group substituted with one or two alkyl, carboxyalkyl, or hydroxyalkyl; or trans The present invention was completed based on the discovery that an iron complex of a compound represented by the formula (a cyclohexene group) or an iron complex of gluconic acid has the above-mentioned remarkable effects. That is, the present invention provides one or two of the above iron complexes.
The present invention relates to a method for removing the influence of ascorbic acid in measuring body fluid components, which is characterized by adding ascorbic acid or more. The compounds represented by (A) above include nitrilotriacetic acid (NTA), dihydroxyethylglycine (DHEG), iminodiacetic acid (IDA), hydroxyethyliminodiacetic acid (HIDA), tris(hydroxymethyl)methylglycine (Tricine ) and nitrilodiacetic acid propionic acid (NDAP), etc.
Compounds represented by (B) include transcyclohexanediaminetetraacetic acid (CyDTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminediacetic acid (EDDA), hydroxyethylethylenediaminetriacetic acid (EDTA-OH), and diaminopropanetetraacetic acid. (Methyl-EDTA), etc.
Iron may be divalent or trivalent, and preferably trivalent iron is used. In addition, oxidizable chromogen systems for quantitative analysis to which the method of the present invention is applied include, for example, 4-aminoantipyrine and naphthol derivatives, 4-aminoantipyrine and aniline derivatives, 4-aminoantipyrine and naphthol derivatives, and many other hydrogen peroxide compounds. There are color-forming systems, and examples of electron transport systems include diaphorase, phenazine methosulfate (PMS), and derivatives thereof. Of course, in addition to these,
The method of the present invention can be applied to any measurement in which ascorbic acid interferes. The method of the present invention is effective when the iron complex is present in the reaction system when measuring body fluid components, so the iron complex may be added to the reaction system by some method. Next, the present invention will be explained using test examples and examples. Test Example 1 In order to investigate the effect of removing the influence of ascorbic acid on iron complexes in hydrogen peroxide system, as a representative example,
NTA, DHEG, IDA, HIDA, Tricine,
NDAP, CyDTA, EDTA, EDDA, EDTA―
2 of each of OH, MeEDTA and gluconic acid
The following tests were conducted using valent and trivalent iron complexes. 1 Reagents used (1) Reaction reagents (see Table 1, 27 types from I to I)
Components of I~Q (1) Buffer solution (Borax Buffer)...Borax 50 mmol/(PH7.0) (2) NTA solution...NTA 0.5 mmol/ (3) NTA-Fe() solution...NTA 0.5 mmol/ Ferrous ammonium sulfate 0.5 mmol/ (4) NTA-Fe() solution...NTA 0.5 mmol/ferric ammonium sulfate 0.5 mmol/ (5) DHEG-Fe() solution...DHEG 0.5 mmol/ferrous ammonium sulfate 0.5 mmol/ (6) DHEG-Fe() solution...DHEG0.5mmol/ferric ammonium sulfate 0.5mmol/ (7) IDA-Fe() solution...IDA0.5mmol/ferrous ammonium sulfate 0.5mmol/ (8) IDA -Fe() solution...IDA0.5mmol/ferric ammonium sulfate 0.5mmol/(9) HIDA-Fe() solution...HIDA0.5mmol/ferrous ammonium sulfate 0.5mmol/(10) HIDA-Fe() solution... HIDA0.5mmol/ Ferric ammonium sulfate 0.5mmol/ (11) Tricine-Fe() solution...Tricine0.5mmol/ Ferrous ammonium sulfate 0.5mmol/ (12) Tricine-Fe() solution...Tricine0.5mmol/ Ferrous sulfate Diferric ammonium 0.5 mmol/ (13) NDAP-Fe() solution...NDAP0.5 mmol/ Ferrous ammonium sulfate 0.5 mmol/ (14) NDAP-Fe() solution...NDAP 0.5 mmol/ Ferric ammonium sulfate 0.5 mmol/ (15) CyDTA-Fe() solution...CyDTA0.5mmol/ ferrous ammonium sulfate 0.5mmol/ (16) CyDTA-Fe() solution...CyDTA0.5mmol/ ferric ammonium sulfate 0.5mmol/ (17) EDTA-Fe () Solution...EDTA0.5mmol/ Ferrous ammonium sulfate 0.5mmol/ (18) EDTA-Fe() solution...EDTA0.5mmol/ Ferric ammonium sulfate 0.5mmol/ (19) EDDA-Fe() solution...EDDA0. 5mmol/ Ferrous ammonium sulfate 0.5mmol/ (20) EDDA-Fe() solution...EDDA0.5mmol/ Ferric ammonium sulfate 0.5mmol/ (21) EDTA-OH-Fe() solution...EDTA-OH0.5mmol/ Ferrous ammonium sulfate 0.5 mmol/ (22) EDTA-OH-Fe()...EDTA-OH0.5 mmol/ferric ammonium sulfate 0.5 mmol/ (23) MeEDTA-Fe() solution...MeEDTA-OH0.5 mmol/Sulfuric acid Ferrous ammonium 0.5 mmol/ (24) MeEDTA-Fe() solution...MeEDTA-0.5 mmol/ferric ammonium sulfate 0.5 mmol/ (25) Gluconate-Fe() solution...gluconate 0.5 mmol/ferric sulfate Ferrous ammonium 0.5 mmol/ (26) Ferrous ammonium sulfate solution... Ferrous ammonium sulfate 0.5 mmol/ [Fe(SO ₄ ) ₂ (NH ₄ ) ₂ ] (27) Ferric ammonium sulfate solution... Ferrous sulfate Iron ammonium 0.5 mmol/ [Fe ₂ (SO ₄ ) ₄ (NH ₄ ) ₂ ] The remainder of each solution above is water.

[Table] 0 marks indicate added items
(2) Coloring solution (PH7.0) Borax 50 mmol / TOOS ^* 3 mmol / 4-aminoantipyrine 1.5 mmol / peroxidase 5000 units / balance water *TOOS: N-ethyl-N-(2-hydroxy-3-sulfopropyl) -m-Toluidine (3) Ascorbic acid solution Ascorbic acid 20 mg/dl Balance water (4) Hydrogen peroxide solution Hydrogen peroxide 1.5 mmol/ Balance water 2 Procedures and results Add (3) to each 2 ml of reaction reagent E in Table 1. ) Add 20μ of ascorbic acid solution or 20μ of purified water,
After heating at 37℃ for 5 minutes, (2) add 1 ml of coloring solution, immediately (4) add 20μ of hydrogen peroxide solution or 20μ of purified water, further heat at 37℃ for 5 minutes, and measure the absorbance at a wavelength of 555nm. It was measured. As a result, as shown in Table 2 below, the influence of ascorbic acid can be completely removed with the reaction reagents of Han to which the iron complex according to the present invention has been added, but with only ferrous ammonium sulfate, the color change The reaction was inhibited, and addition of ferric ammonium sulfate and NTA alone was not effective in removing the effect of ascorbic acid.

【table】

[Table] Test Example 2 In order to investigate the effect of removing the influence of ascorbic acid on iron complexes in formazan system, as a representative example,
Using CyDTA-Fe() and CyDTA-Fe(),
The following tests were conducted. 1 Reagents used (1) Reaction reagents (see Table 3 A to F6 types) A to
Components of F (1) Buffer solution...borax 50 mmol/(PH7.0) (2) CyDTA solution...CyDTA 0.5 mmol/ (3) CyDTA-Fe() solution...CyDTA 0.5 mmol/ferric ammonium sulfate 0.5 mmol/ (4) CyDTA-Fe() solution...CyDTA0.5 mmol/ Ferrous ammonium sulfate 0.5 mmol/ (5) Ferrous ammonium sulfate solution Ferrous ammonium sulfate 0.5 mmol/ (6) Ferrous ammonium sulfate solution ...Ferrous ammonium sulfate 0.5 mmol/The remainder of each solution above is water.

[Table] 0 marks indicate added items
(2) Coloring liquid (PH7.0) Borax 50mmol/Phenazine methosulfate
0.3mmol/ Nitrotetrazolium blue
1.5 mmol/Remaining water (3) Ascorbic acid solution Ascorbic acid 20 mg/dl Remaining water (4) NADH solution reduced nicotinamide adenine dinucleotide 2 mmol/Remaining water 2 Procedures and results 2 ml each of reaction reagents A to F in Table 3 Add (3) 20μ of ascorbic acid solution or 20μ of purified water, heat at 37℃ for 5 minutes, (2) add 1ml of coloring solution, and immediately add (4) 20μ of NADH solution or purified water.
Add 20μ and further heat at 37℃ for 5 minutes.
Absorbance at a wavelength of 580 nm was measured. As a result, as shown in Table 4 below, when the CyDTA-iron complex according to the present invention was added (C,
D) The influence of ascorbic acid in the formazan system can be completely removed, but addition of ferric ammonium sulfate, ferrous ammonium sulfate, or CyDTA alone had no effect.

[Table] Example 1 Neutral fat measurement 1 Reagents (1) Iron complex addition reagent PH7.6 Tris(hydroxymethyl)aminomethane 0.1mol/TOOS 2mmol/Glycerokinase 100u/Glycero-3-phosphate oxidase
10000u/ Peroxidase 2500u/ Adenosine triphosphate 1mmol/ EDDA-Fe() 0.1mmol/ Remaining water (2) Control reagent PH7.6 Reagent from (1) minus EDDA-Fe() (3) Reaction reagent PH7 .6 Tris(hydroxymethyl)aminomethane 0.1mol/Lipoprotein lipase 200000u/4-aminoantipyrine 3mmol/Remaining water (4) Ascorbic acid solution Ascorbic acid 50mg/dl Remaining water 2 Procedures and results Added 20μ of serum (sample) Prepare two test tubes and one test tube containing 20μ of purified water (reagent blank), and add (4) ascorbic acid solution 20μ to one of the former, and purified water to the other.
Add another 20μ. After adding 2 ml of (1) iron complex addition reagent to each of these three test tubes,
The mixture was heated at 37° C. for 5 minutes, then 1 ml of reaction reagent (3) was added, heated at 37° C. for 5 minutes, and the absorbance at a wavelength of 555 nm was measured. In the same manner, (1) the iron complex-added reagent was replaced with (2) the control reagent, and the absorbance was measured. The results are shown in Table 5, and the use of the iron complex-added reagent was very effective in eliminating the effects of ascorbic acid.

[Table] Example 2 Measurement of transaminase (1) 1 Reagent (1) Reagent PH for iron complex-added GOT (GOT: glutamate oxacetate transaminase)
7.4 Potassium phosphate 10mmol / TOOS 2mmol / L-aspartic acid 100mmol / α-ketoglutaric acid 5mmol / Thiamine pyrophosphate 0.4mmol / Oxaloacetate decarboxylase 2000u / Pyruvate oxidase 3000u / Peroxidase 2000u / DHEG-Fe() 0.5mmol / Remaining water (2) Control GOT reagent PH7.4 (1) without DHEG-Fe() (3) Iron complex-added GPT reagent PH7.4 Phosphate-potassium 10 mmol/TOOS 2 mmol/L - Alanine 100mmol / α-ketoglutaric acid 5mmol / Thiamine pyrophosphate 0.4mmol / Pyruvate oxidase 3000u / Peroxidase 2000u / DHEG-Fe() 0.5mmol / Balance water (4) Control GPT (GPT: glutamate transaminase) reagent (3) (5) Reaction reagent 4-aminoantipyrine 2.5 mmol/Remaining water (6) Reaction stop solution Sodium citrate 0.1 mol/Remaining water (7) Ascorbic acid solution Ascorbic acid 20 mg/ dl Remaining water 2 Procedure and results Prepare two test tubes containing 20μ of serum (sample) and one test tube containing 20μ of purified water (reagent blank), one of the former containing (7) ascorbic acid. 20μ of solution and purified water in the other bottle
Add another 20μ. After adding 0.8 ml of (1) iron complex-added GOT reagent to each of these three test tubes, the mixture was heated at 37°C for 3 minutes, and then (5) reaction reagent
After adding 0.2 ml and reacting at 37℃ for 20 minutes, (6)
2 ml of reaction stop solution was added, and the absorbance at a wavelength of 555 nm was measured. In the same way, use the reagent in (1) as a control in (2).
The absorbance was measured in place of the GOT reagent, (3) iron complex-added GPT reagent, or (4) control GPT reagent. The results are shown in Table 6, where the addition of the iron complex addition reagent was highly effective in eliminating the effects of ascorbic acid.

[Table] Example 3 Measurement of transaminase (2) 1 Reagent (1) Reagent for GOT with iron complex addition PH7.4 Potassium phosphate 10 mmol / 4-aminoantipyrine 0.5 mmol / 2.4-dichlorophenol 2 mmol / L-aspartic acid 100mmol / α-ketoglutaric acid 5mmol / Thiamine pyrophosphate 0.4mmol / Oxaloacetate decarboxylase 2000u / Pyruvate oxidase 3000u / Peroxidase 2000u / EDTA-OH-Fe() 0.2mmol / Remaining water (2) Control reagent for GOT PH7. 4 Reagent from (1) minus EDTA-OH-Fe() (3) Iron complex-added GPT reagent PH7.4 Potassium phosphate 10 mmol/4-aminoantipyrine 0.5 mmol/2,4-dichlorophenol
2 mmol/ L-alanine 100 mmol/ α-ketoglutaric acid 5 mmol/ Thiamine pyrophosphate 0.4 mmol/ Pyruvate oxidase 3000u/ Peroxidase 2000u/ EDTA―OH―Fe() 0.2 mmol/ Balance water (4) Control GPT reagent PH7.4 ( 3) from the GPT reagent EDTA-OH-Fe
(5) Reaction stop solution Sodium citrate 0.1 mmol/Remaining water (6) Ascorbic acid solution Ascorbic acid 10 mg/dl Remaining water 2 Procedures and results Two test tubes containing 20μ of serum (sample) and Prepare one test tube containing 20μ of purified water (reagent blank), and add 20μ of (6) ascorbic acid solution to one of the former tubes, and purified water to the other.
Add 20μ. After adding 1 ml of the reagent (1) to each of these three test tubes and heating at 37°C for 20 minutes, (5) 2 ml of the reaction stop solution was added, and the absorbance at a wavelength of 500 nm was measured. Similarly, use the reagent (1).
Absorbance was measured in place of (2) control GOT reagent, (3) iron complex-added GPT reagent, or (4) control GPT reagent. As shown in Table 7, the results revealed that the use of the iron complex-added reagent was highly effective in eliminating the effects of ascorbic acid.

[Table] Example 4 Measurement of lactate dehydrogenase 1 Reagents (1) Iron complex addition reagent PH8.6 Tris(hydroxymethyl)aminomethane 0.1 mmol/DL-lactic acid 0.15 mmol/β-nicotinamide adenine dinucleotide 5 mmol/Diaphorase 3000u/ CyDTA-Fe() 1.5mmol/Remaining water (2) Control reagent PH8.6 Reagent from (1) except CvDTA-Fe() (3) Coloring liquid Nitrotetrazolium blue
3 mmol/Remaining water (4) Reaction stop solution Hydrochloric acid 0.1 mmol/Remaining water (5) Ascorbic acid solution Ascorbic acid 20 mg/dl Preparation water 2 Procedure and results 2 test tubes containing 20μ of serum (sample) and purified water Prepare one test tube containing 20μ (reagent blank), and add (5) ascorbic acid solution 20μ to one of the former, and purified water to the other.
Add 20μ. Add 0.8 ml of the reagent (1) to each of these three test tubes and heat at 37°C for 3 minutes.
(3) After adding 0.2 ml of the coloring solution and reacting at 37°C for 20 minutes, (5) adding 5 ml of the reaction stop solution, the absorbance at a wavelength of 570 nm was measured. The same procedure was carried out by replacing the reagent (1) with the reagent (2), and the absorbance was measured. As shown in Table 8, the use of iron complexes was highly effective in eliminating the effects of ascorbic acid.

[Table] As is clear from the above, the present invention adds the above-mentioned iron complex and measures body fluid components.
It is possible to completely eliminate the influence of ascorbic acid on measured values.

Claims (1)

[Claims] 1. The following general formula (A) or (B) (In the formula, R ₁ to ₅ are each H; an alkyl group; a carboxyalkyl group; or a hydroxyalkyl group, and X is an ethylene group; an ethylene group substituted with one or two alkyl, carboxyalkyl, or hydroxyalkyl; or 1. A method for eliminating ascorbic acid in measuring body fluid components, which comprises adding one or more divalent or trivalent iron complexes of the compound represented by (a transcyclohexene group). 2 Divalent or trivalent iron complex of gluconic acid 1
1. A method for eliminating ascorbic acid in measuring body fluid components, the method comprising adding a species or two or more species.