JPH07265097A - Determination of iron - Google Patents

Abstract

PURPOSE:To enable high-sensitivity determination of iron under mild conditions simply in a shortened time by generating hydrogen peroxide through an enzyme reaction, oxidizing divalent iron into trivalent iron with the hydrogen peroxide in the presence of a reducing agent and determining the corresponding change in the hydrogen peroxide. CONSTITUTION:An aqueous chelate such as EDTA or the like is added to a sample from living body to release iron from iron-binding proteins such as transferrin, albumen or globulin. Then, 4-aminoantipyrine, ascorbic acid as a reducing agent and glucose oxidase are added, then glucose and peroxidase are admixed thereto to liberate hydrogen peroxide so that the divalent iron is oxidized into the trivalent iron by the hydrogen peroxide in the presence of a reducing agent. Then, the change in hydrogen peroxide corresponding to the iron oxidation is determined with the peroxidase-4-aminoantipyrin system whereby high-sensitive determination of iron in a sample can be done.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an extremely sensitive iron quantification method utilizing the fact that hydrogen peroxide generated by an enzymatic reaction is amplified and consumed by divalent iron ions in the presence of a reducing agent.

[0002]

2. Description of the Related Art Bipyridine, 0-phenanthroline, bathophenanthroline,
There is a colorimetric method using tripyridyltriamine, thioglycolic acid, tyrone and the like. However, these methods are not suitable for simple and rapid measurement such as enzyme reaction because they utilize organic solvent extraction and the reaction and color development conditions are not mild. As a method for measuring iron ion using an enzymatic reaction, a method utilizing activation of aconitase by iron ion has been reported (32nd Annual Meeting of the Japan Society for Clinical Chemistry, p56b).

It is well known that divalent iron ions reduce hydrogen peroxide, and this can be used to determine divalent iron ions. There is a method of measuring iron electrochemically, and it has long been known as coulometry for iron. However, these methods have poor sensitivity and are not suitable for the analysis of trace amounts of iron. In addition to these methods, invention of a method for detecting iron with high sensitivity is required.

[0004]

[Problems to be Solved by the Invention]
In order to detect a small amount of iron ions such as pt (ng / ml), a method of detecting the change amount of a small amount of hydrogen peroxide corresponding to the amplification of hydrogen peroxide reducing action by divalent iron ions with high sensitivity is used. There must be.

[0005]

The present invention is directed to an aqueous medium,
Hydrogen peroxide is generated by an enzymatic reaction or an electrochemical method, and hydrogen peroxide generated in the coexistence of a reducing agent is reacted with divalent iron. It is characterized in that it is detected by a dynamic method. Since the iron concentration targeted by the present invention is extremely small, the amount of hydrogen peroxide consumed by divalent iron is too small to be detected by an electrochemical method or an absorptiometric method. It is invalid as a method. The present invention is based on the finding that hydrogen peroxide can be synergistically consumed to coexist divalent iron with a small amount of a reducing agent, and iron can be quantified with high sensitivity.

Here, the aqueous medium means a liquid containing water such as a buffer solution and physiological saline, and the buffer solution is tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution, phosphate buffer solution, Good buffer solution. A typical example is barbital buffer. As the iron reducing agent, one that is lower than the apparent redox potential of the Fe ²⁺ —Fe ³⁺ system is used. Examples include ascorbic acid, hydroxylamine hydrochloride, sodium thiosulfate, thioglycolic acid, hydroquinone, and hydrazine sulfate. Particularly, hydroxylamine and its salt, and hydrazine and its salt are excellent reducing agents because they do not inhibit the peroxidase reaction and the consumption reaction of hydrogen peroxide is slow. Since ascorbic acid can be consumed by ascorbic acid oxidase, most of it can be consumed before the hydrogen peroxide generation-coloring reaction. Incidentally, hydroxylamine can also be eliminated by hydroxylamine dehydrogenase. It is also possible to use, as a reductant, a ferrocyanion ion produced by a diaphorase reaction from a nicotinamide adenine dinucleotide reductant (NADH) and ferricyan ion. In this case, the hydrogen peroxide consumption rate of ferrocyan ion is slow.

As the hydrogen peroxide producing reaction, there is an electrochemical method, but the oxidase enzymatic reaction which produces hydrogen peroxide by the substrate and oxygen is most suitable. In this case, it does not directly produce hydrogen peroxide, but includes all enzymatic reactions that are ultimately coupled to the oxidase enzymatic reaction.
Among the oxidase enzymatic reactions, as a method that is often used with a substance contained in a biological sample or the like as a substrate, there are a glucose oxidase method, a cholesterol oxidase method, a galactose oxidase method, a glycerol oxidase method, a urate oxidase (uricase) method and the like. Although not generally used, amino acid oxidase method, amine oxidase method, aldehyde oxidase method, glutamine oxidase method, glycol oxidase method, dihydroorotic acid oxidase method, pyruvate oxidase method, hexose oxidase method, monoamine oxidase method, latosterol oxidase method, The lysine oxidase method, the oxalate oxidase method, the hydroxy acid oxidase method and the like are available.

[0008] As a substance that does not exist or is present in a trace amount in a biological sample and both the substrate and the enzyme are stable for a long period of time,
There are a choline oxidase method, a glycerol-3-phosphate oxidase method, and a xanthine oxidase method.

When the substrate is present in the sample in advance, the substrate is erased by another enzymatic reaction in advance and changed to another one, and then the above-mentioned hydrogen peroxide production reaction is carried out or a large amount of the substrate is present. Then, there is a method of eliminating the influence of the increase of the substrate contained in the sample. These methods are well established and well used in enzymatic assays. Some of the above-mentioned enzymatic reactions for producing hydrogen peroxide are inhibited by Cu ²⁺ , but are not inhibited by Fe ²⁺ in a small amount.

To detect the produced hydrogen peroxide with high sensitivity, an electrochemical method [Shuichi Suzuki, edited by Ion and Enzyme Electrodes, p86-106, Kodansha Scientific]
Besides, there are oxidase detection reaction (catalase conjugated system, peroxidase conjugated system), chemiluminescence method and the like. The catalase coupling system is a method utilizing the Hantzsch reaction, 4-
Amino-3-hydrazino-5-mercapto-1,2,4
-There is a method using triazole or 3-methyl-2-benzothiazoline hydrazine. For the peroxidase-conjugated system, many high-sensitivity color reagents have been developed. These are ["Clinical Chemistry Practice Manual", Examination and Technology Special Issue,
1993, 21st volume, p23, medical bookstore]] as a list. The molar extinction coefficient of these coloring reagents is 1 × 10 ⁴ to 8 × 10 ⁴ , and 4-aminoantipyrine having the smallest molar extinction coefficient (molar extinction coefficient 1 × 10 4
⁴ ) Even with the reagent, 5 ppb (10 ng / ml) of iron can be measured with sufficient sensitivity. For N-sulfopropylamine, which has a large molar extinction coefficient, ppt (pg / m
The iron of 1) can be measured sufficiently. It is possible to quantify a trace amount of iron by combining a coloring reagent having such a large molar extinction coefficient with a hydrogen peroxide consuming reaction by Fe ²⁺ coexisting with a reducing agent. In addition to the Trinder color-developing reagent as described above, a redox color-developing reagent can also be used, but since it is affected by a reducing agent that reduces Fe ³⁺ to Fe ²⁺ , it can be used after the iron reducing agent is eliminated in advance. . This reagent is described in Dojin Chemical Research Institute Catalog DOJIN17, p10. Although less sensitive, the reaction of iodine ions or ferricyan ions with hydrogen peroxide can also be used. In addition to the spectroscopic method as described above, the hydrogen peroxide electrode method and the peroxidase-immobilized enzyme electrode method can be used as the highly sensitive detection method.

In order to accurately detect the amount of hydrogen peroxide consumed by Fe ²⁺ and its change, the amount of hydrogen peroxide must be appropriate for the detection reaction. In the present invention, a method of generating hydrogen peroxide by an oxidase enzymatic reaction is adopted. According to this,
It is said that it can be adjusted in any way by controlling the coenzyme, the substrate mass, etc., and that the hydrogen peroxide production rate can also be adjusted by controlling the enzyme amount, coenzyme amount, activator amount, etc. It has excellent advantages.

Next, when iron is bound to a protein (transferrin, albumin, etc.) as in a biological sample, the iron is released from the protein using a chelating agent. In the present invention, a method using a chelating agent is an important constituent element for such a sample. This is because, in a method for quantifying iron using an enzyme having iron as a molecular component (prosthetic factor), the addition of such a chelating agent deactivates and the reaction does not proceed, but in the oxidase detection reaction used in the present invention, Since iron is not used as a coenzyme and is not deactivated, a chelating agent can coexist. By coupling a chelating agent, the process of acidifying pH and deproteinizing, which are conventionally required to release iron from protein, is no longer necessary, greatly simplifying the measurement operation and freeing the measurement system. Various combinations are possible. Furthermore, an important point is that the oxidoreductase reaction such as the oxidase detection reaction of the present invention was found to proceed similarly to the free divalent iron ion even in the presence of the divalent iron chelate. By utilizing this phenomenon, it becomes possible to quantify iron in serum by oxyreductase reaction other than oxidase reaction. Such enzyme reactions include ferrooxidase I, ferrooxidase II, and lipoamide reductase.
Similarly, oxidoreductase, which is inhibited by divalent iron ion, can also be quantified by an inhibition method in the presence of a chelating agent, and examples thereof include choline dehydrogenase.

As the iron chelating agent, ethylenediaminetetraacetic acid, trans-1,2-cyclohexanediamine-
N, N, N ', N'-tetraacetic acid, diethylenediaminepentaacetic acid, glycoletherdiaminetetraacetic acid, triethylenetetraminehexaacetic acid, diaminopropanoltetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diaminopropanetetraacetic acid, etc. are stability constants. Is greatly preferable. Nitrotriacetic acid, dihydroxyethylglycine, iminotriacetic acid, hydroxyethyliminodiacetic acid, ethylenediaminedipropionic acid and the like can be used although their stability is slightly small. Not only such a chelating reagent, but also a water-soluble iron-binding reagent such as sodium bathophenanthrin sulfonate and feron can be used.

The essence of the embodiment of the present invention will be described below. The embodiment of the present invention comprises two steps of hydrogen peroxide production process and detection process. Samples include tap water, waste water,
It can be applied to a wide range of biological samples (blood, serum, plasma, urine, hemoglobin, etc.) and foods. First, 0.01 to 1 mg / ml of a reducing agent was added to an aqueous medium, then a sample containing iron was added, and the mixture was incubated at 8 to 50 ° C for 0 to 5 minutes (in the case of ascorbic acid and hydroxylamine hydrochloride, the reduction was instantaneous. (Incubation is not necessary). Reduce ferrous iron to ferrous iron. In the case of only divalent iron, the reducing agent may be added later. When the method of producing hydrogen peroxide by the oxidase reaction is used, the oxidase or its substrate may coexist in the aqueous medium at this time.
For example, 0.1 to 10 mg / ml of choline chloride or the like is added. Also, a substrate for color reaction may be added. For example, 0.05 to 1 mg / m of 4-aminoantipyrine, etc.
1 is added. Next, an aqueous medium containing the remaining substrate, the enzyme for the oxidase reaction and the enzyme for the color development reaction is added to the above liquid, the amount of change in absorbance produced is determined, and the amount of iron is determined. 0.2 to 20 U / ml is used as the oxidase enzyme, and 0.1 to 20 U / ml of peroxidase is used as the color development reaction. The amount of this enzyme depends on the kind of the reducing agent, and may be a small amount in the case of hydroxylamine hydrochloride and hydrazine sulfate.
The remaining substrate is N-ethyl-N- (3-methylphenyl) -N'-succinylethylenediamine 0.1-
Add 2 mg / ml. The hydrogen peroxide production and color reaction are carried out at pH 6 to 7.5 and temperature 8 to 50 ° C. which are suitable conditions for enzymatic reaction.

Regarding the pH of the aqueous medium, it is preferable that the reduction of ferric iron to ferrous iron is made acidic so as to prevent ferrous iron from being oxidized to trivalent iron by oxygen. In this case, an aqueous medium having a weak buffering action (water may be used alone) is used, and a strong buffer solution in the next reaction process is used so that the pH becomes appropriate for the reaction. When the process of producing and detecting hydrogen peroxide is carried out by an enzymatic reaction, the pH of the buffer solution is set to 5 to 8, particularly pH 6.0.
7.5 is desirable.

According to the literature, ascorbic acid is 6 mg / d
It is desirable to add it at a ratio of 1 and the oxidase reaction is carried out after reducing the ferric iron. Ascorbic acid develops color at the same time as ascorbic acid is eliminated by adding ascorbic acid oxidase to the color reaction system. In this case, a small amount of ascorbic acid remaining as a reducing agent exerts a synergistic effect with divalent iron.

In the oxidase reaction, there are cases where the detection process is carried out after a certain amount of hydrogen peroxide is generated, and cases where both are carried out simultaneously. The former has an epoch-making advantage that iron can be accurately measured even if a large amount of hydrogen peroxide consuming substances coexist in the sample. That is, even if a large amount of reducing substances such as ascorbic acid, uric acid, and bilirubin are present in the sample, if a series of reactions are performed without adding an iron reducing agent, iron does not participate in the reaction and only the blank is left. Is quantified. Next, a reducing agent for iron is added to carry out a series of reactions, and the two can be subtracted to determine iron. This method is extremely useful for samples in which iron is present as trivalent iron due to the enzymatic action even in the presence of ascorbic acid and the like such as serum and plasma.

With respect to the biological sample, when the substrate in the oxidase reaction is contained in the sample, it is necessary to carry out the elimination reaction. For example, when glucose oxidase is used, endogenous glucose is eliminated with glucose oxidase or hexokinase is eliminated. When hexokinase is used, since it competes with the oxidase reaction, it is necessary to add an inhibitor such as a magnesium chelating agent to inactivate it. Since many of the iron chelating agents in the present invention are also magnesium chelates,
It can be said that the use of a chelating agent is an extremely excellent method.

The chelating agent for separating iron from protein may be coexistent with the oxidase reaction or may be conjugated during the next oxidase detection reaction. In the latter case, iron may be separated from transferrin, albumin or the like in advance. For this purpose, the pH of the liquid is 2-4.

According to the present invention, the iron-binding ability of serum, which has been difficult by other methods and other enzymatic methods, can be easily measured. The principle is shown below. First, according to the method described above, serum iron is quantified in the presence of an iron chelating agent. Next, an excessive amount of iron ion is added to serum to quantify iron without the coexistence of an iron chelating agent. In this case, the protein is saturated with iron,
Since that portion does not react, only free iron ions are measured. The iron binding capacity can be obtained by subtracting the amounts of serum iron and free iron from the added iron amount.

In the present invention, when ascorbic acid or the like, which is a strong reducing agent, is used, it is slightly affected by copper. Regarding copper, it is advisable to calculate and add the same amount of copper nitrate as the copper in the sample. Alternatively, a reagent such as bathocuproin that specifically causes a precipitate with copper is added. The reducing substances of uric acid and bilirubin undergo color reaction after being erased by urate oxidase and bilirubin oxidase, respectively. In this case, hydrogen peroxide generated by the erasing reaction disappears by the iron reducing agent and does not affect the color forming reaction.

[0022]

【Example】

Example 1 Method Using Glucose Oxidase and Ascorbic Acid (1) Preparation of Standard Solution for Iron Calibration Curve Ferric sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with distilled water, and a drop of hydrochloric acid was added, followed by constant volume. 1, 2.5, 5, 10 μg / ml
A standard solution for iron (III) calibration was prepared. However, separately, the concentration was measured by an atomic absorption spectrophotometric method using an iron standard solution manufactured by Wako Pure Chemical Industries as a control to obtain a correction coefficient. (2) Determination of iron 0.10 ml of a standard solution for iron calibration is placed in a test tube, 4-aminoantipyrine 0.1 mg / ml, and ascorbic acid 0.
08 mg / ml, and glucose oxidase (Asp
ergillus niger, manufactured by Wako Pure Chemical Industries, Ltd.) 30
25mM acetate buffer (pH 5) containing U / ml 1.0m
1 and incubate at 37 ° C. for 5 minutes. Then N
-Ethyl-N- (3-methylphenyl) -N'-succinylethylenediamine (EMSE) 0.6 mg / ml,
100 mM Good buffer solution containing 0.05 mg / ml glucose, 10 U / ml peroxidase (derived from horseradish, manufactured by Wako Pure Chemical Industries) and 10 U / ml ascorbate oxidase (derived from cucumber, manufactured by Wako Pure Chemical Industries) (HE
2.0 ml of PES, pH 6.8) previously incubated at 37 ° C. was added to the above solution, and the change in absorbance was measured with a spectrophotometer (UV3400 manufactured by Hitachi). The calibration curve obtained is shown in FIG. In this method, even if the sample contains a small amount of glucose, it is decomposed by glucose oxidase into hydrogen peroxide, which is further reduced by ascorbic acid and does not affect the color development. However, when glucose becomes a considerable amount, ascorbic acid becomes insufficient, which causes an error. Further, when the sample contains a large amount of ascorbic acid, it also affects the color development reaction. The more ascorbate oxidase, the more accurate the result.

[0023]

Example 2 Method Using Choline Oxidase and Ascorbic Acid (1) Preparation of Standard Solution for Iron Calibration Curve In the same manner as in Example 1, 125, 250, 375,
475 μg / dl was prepared. (2) Quantification of iron Take 0.080 ml of the standard solution for iron calibration curve in a test tube, and add 0.1 mg / ml of 4-aminoantipyrine, 0.4 mg / ml of choline chloride, and 0.12 mg / m of ascorbic acid.
1.0 ml of 20 mM acetate buffer (pH 2) containing 1 is added and incubated at 37 ° C. for 5 minutes. Next, EMSE
0.6 mg / dl, peroxidase (derived from horseradish, manufactured by Oriental Yeast) 10 U / ml, choline oxidase (derived from Alcaligenes, manufactured by Wako Pure Chemical Industries) 6 U /
ml, ascorbate oxidase 8 cucumber origin, manufactured by Wako Pure Chemical Industries, Ltd.) 200 mM Good buffer solution (pH 7.0, 25 ° C.) containing 3 U / ml incubated at 37 ° C. 2 ml was added to the above solution, and 1 minute later And the absorbance after 3 minutes was measured with a spectrophotometer (Hitachi UV3400),
Both absorbance differences were calculated. The calibration curve obtained is shown in FIG. In this method, a solution having a pH of 2 is mixed with a sample and incubated at 37 ° C. for 5 minutes, whereby colloidal iron hydroxide and colloidal iron oxide can be decomposed and quantified.
Although this method is an end point method, the rate method can be performed by using choline chloride in a multiple amount. The end point method or the rate method depends on choline chloride and the amount of choline oxidase.
The reason why a large amount of ascorbic acid was added was to make the absorbance after 0.5 minutes a blank test value. For clean samples, a small amount of ascorbic acid is sufficient.

[0024]

Example 3 Method Using Glycerol-3-Phosphate Oxidase and Hydroxylamine Hydrochloride (1) Preparation of Standard Solution for Iron Calibration Curve Ferrous sulfate of reagent grade ferrous sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was made constant with distilled water. , 125, 250, 375 μg / dl iron (II) standard curve standard solution was prepared. (2) Quantification of iron Take 0.030 ml of the standard solution for iron calibration curve in a test tube, and add 0.3 mg / ml of 4-aminoantipyrine, 0.15 mg / ml of hydroxyamine hydrochloride, and glycerol-3-
Aqueous solution containing 1.5 mg / ml phosphoric acid (pH 2.5, 2
(5 ° C) 1.0 ml, and incubate at 37 ° C.
Next, EMSE 0.3 mg / ml, peroxidase (derived from horseradish, manufactured by Wako Pure Chemical Industries, Ltd.) 0.4 U / ml,
Glycerol-3-phosphate oxidase (derived from microorganism, EC 1.1.321, manufactured by Boehringer Mannheim) 1.5 U / ml, 200 mM Good buffer solution (pH 7.5, 25 ° C) containing sodium ethylenediaminetetraacetate 5 mg / ml ) 2.0 ml in advance at 37 ° C
What was incubated in the above was added to the above solution, and the resulting change in absorbance (0.2 to 0.7 / 0.5 minutes) was measured by a spectrophotometer (UV3400 manufactured by Hitachi). The calibration curve is shown in FIG. Next, 0.030 mL of serum was taken and measured in the same manner. As a result, the measured value was 95 mg / dl. The result of this serum measured by the bathophenanthroline absorptiometry was 92 mg / ml, which was in very good agreement.

[0025]

Example 4 Using Hydrazine Sulfate (1) Preparation of Standard Solution for Iron Calibration Curve The same as in Example 3, but with concentration series of 2.4, 6.0,
It was set to 12, 25 μg / ml. (2) Quantification of iron 0.10 ml of a standard solution for iron calibration curve was placed in a test tube, and 4-aminoantipyrine 0.2 mg / ml, choline chloride 0.6 mg / ml, and hydrazine sulfate saturated water (20 ° C).
Aqueous solution containing 0.04 ml / ml (pH 2.5, 25
(C) 2.0 ml, and incubate at 25 C for 5 minutes. Next, EMSE 1 mg / ml, peroxidase (derived from horseradish, manufactured by Wako Pure Chemical Industries, Ltd.) 0.5 U / ml,
200 mM Good (HEP) containing 1.1 U / ml of choline oxidase (derived from Alcaligenes, manufactured by Wako Pure Chemical Industries, Ltd.)
ES) buffer (pH 7.0, 25 ° C.) incubated at 25 ° C. 1 ml was added to the above solution, 0.5
Spectrophotometer (Hitachi UV)
3400), and the absorbance difference between both was calculated. The calibration curve obtained is shown in FIG.

[0026]

The effects of the present invention are as follows. Since the present invention uses an enzymatic reaction, iron can be easily measured in a short time under mild reaction conditions. In addition, it is possible to generate an appropriate amount of hydrogen peroxide in the same solution as the detection by an enzymatic reaction, and the iron concentration during the reaction is ppt.
An extremely high sensitivity of (ng / ml to pg / ml) can be obtained. As the concentration in the sample, several ppb (several μg / dl) giving 0.001 change in absorbance can be detected from FIG. Since the enzyme used in the present invention does not use iron as a coenzyme, it is possible to coexist with an iron chelating agent, and it is possible to quantify iron in a milder and simpler process than the method using iron as a coenzyme. Taking advantage of this feature, the iron-binding ability in serum, which has been difficult with other methods and other enzyme methods, can be easily measured. In the method of quantifying iron by oxidizing divalent iron into trivalent iron by potentiostatic coulometry, the amplification effect of the present invention enables highly sensitive quantification by coexisting with a reducing agent. The principle of the present invention can be applied to the determination of trace amounts of redox metals such as copper and manganese.

[0027]

[Brief description of drawings]

FIG. 1 Calibration curve of iron when glucose oxidase is used

FIG. 2 Calibration curve of iron using choline oxidase

FIG. 3 Calibration curve of iron using glycerol-3-phosphate oxidase

FIG. 4 Calibration curve of iron when using hydrazine sulfate

Claims (5)

[Claims] 1. An iron analysis method which comprises oxidizing divalent iron to trivalent iron in the presence of a reducing agent and quantifying the corresponding change. 2. A method for producing a hydrogen peroxide, oxidizing divalent iron to trivalent iron with hydrogen peroxide in the presence of a reducing agent, and quantifying a corresponding change in the amount of hydrogen peroxide. 1
Method for iron described in 3. The iron analysis method according to claim 2, wherein the hydrogen peroxide generation method is an oxidase enzymatic reaction, and the hydrogen peroxide quantification method is an oxidase detection reaction. 4. The iron analysis method according to claim 2, wherein an iron chelating agent is allowed to coexist. 5. A method for quantitatively determining iron, which comprises releasing iron from an iron-binding protein such as transferrin, albumin, or globulin using an aqueous chelating agent to carry out an oxidoreductase enzymatic reaction.