JPH06153993A - Method for determining serum iron and determining composition - Google Patents

Abstract

PURPOSE:To accurately and efficiently determine serum iron even in a very small amount in simple operation without any measuring mistake by isolating iron from transferrin in serine and measuring aconitase activity while binding the isolated iron to aconitase. CONSTITUTION:A buffer solution of acidic pH is added to serum to isolate iron from transferase in serum and this isolated iron is bound to aconitase by action of a reducing agent such as ascorbic acid on the isolated iron to form an active type aconitase. Sodium citrate is converted into isocitric acid by this active type aconitase and this isocitric acid is made to react with isocitric acid dehydrogenase using nicotinamide adenine dinucleotide phosphates(NAD) or/and nicotinamide adenine dinucleotide phosphates(NADP) as coenzymes and the serum iron is accurately and effectively determined even in very small amount from amounts of these coenzymes converted to reduction type by measurement of 340nm absorption due to spectrophotometer.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying serum iron, and more specifically, it releases affixed iron to transferrin and exhibits no activity unless iron is added (EC 4.2. 1.3) are brought into contact with each other to form an active form, and citric acid generated from citric acid is converted to isocitrate dehydrogenase (EC 1.1.1.41, EC 1.1.41).
The present invention relates to an enzyme quantification method and a reagent for measurement, which have high sensitivity for quantifying serum iron amount by the reaction of 1.1.42) and have a reduced number of steps, and which are highly effective.

[0002]

2. Description of the Related Art The total amount of iron in the body is about 4 g, 2/3 of which is 1 / the remaining amount in erythrocyte hemoglobin.
3 is stored as iron in the liver, spleen, bone marrow and other tissues. The total amount of serum iron is 3 to 4 mg, and
-Binds to transferrin belonging to globulin (part 1)
The molecule exists by binding two iron atoms. The serum iron concentration is
Dependent on the extent of erythrocyte production and destruction, decreased hematopoiesis in the bone marrow results in stagnation of serum iron flow, which increases serum iron levels and vice versa. From this, it is considered that the serum iron concentration reflects the function of the hematopoietic organ. In addition, the dynamics of stored iron are also related to serum iron, and in the case of hepatitis and the like, stored iron is mobilized from the liver and moved to other tissues, so that serum iron increases. As mentioned above, serum iron is not only associated with blood diseases (iron deficiency anemia, aplastic anemia, pernicious anemia, hemolytic anemia, leukemia, polycythemia vera, etc.), but also with various diseases (infection, acute hepatitis, cirrhosis). , Hemochromatosis, nephrosis, etc.), and its measurement is regarded as important in clinical tests.

Methods for quantifying serum iron include Matsubara method, international standard method, autoanalyzer method, atomic absorption method and the like. The principle of the Matsubara method and the international standard method is to release iron with an acid, deproteinize it, reduce iron with a reducing agent, and develop color with a coloring agent. However, the molar extinction coefficient of BPT is small and the sensitivity is high. Since it is low, a large amount of serum is required, it is susceptible to interference, and it is difficult to process multiple samples because it is a manual method. The autoanalyzer method is a simple automation of the principles of the Matsubara method and the international standard method, so the sensitivity problem has not been solved at all and interference with hemoglobin, bilirubin, etc. is observed. Atomic absorption method requires pretreatment, and there are considerable variations in measured values due to differences in treatment methods.There are many problems such as poor sensitivity and the need for expensive special equipment. I have.

[0004]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned drawbacks of the conventional methods all at once, and accurately and accurately determines the amount of iron in a living body, particularly in serum without the need for skill. Capable of measuring, enabling automation or multi-sample processing. The purpose is to develop a new highly sensitive assay system for serum iron.

[0005]

The present invention has been made in order to achieve the above-mentioned object, and the present inventors have found that in order to quickly and accurately determine the amount of serum iron, iron As a result of detailed examination of the relationship between the enzyme activity and various enzyme activities, aconitase requires iron in order to express the enzyme activity,
Knowing that the activity of aconitase changes in response to an increase in serum iron concentration, and confirming that the serum iron amount can be measured by measuring the change, further examination results,
The present invention has been completed.

It is well known that aconitase requires iron for its activity expression [protein nucleic acid enzyme Vol.
22 No. 12 1293-1302 (1977);
THE JOURNAL OF BIOLOGICAL
CHEMISTRY Vol. 236, No. 1
2, December 1961; THE JOUR
NAL OF BIOLOGICAL CHEMIST
RY Vol. 242, No. 8, Issue o
f April 25, pp. 1870-1879,
1967; THE JOURNAL OF BIOL
OGICAL CHEMISTRY Vol. 246,
No. 3, Issue of February 1
0, pp. 772-779, 1971; Cell,
Vol. 64, 881-883, March
8, 1991]. However, in all the literature, for example, low concentrations of iron such as serum iron (normal value; serum male 1
1.6-34.9 μM, female; 7.29-29.5 μ
There is nothing that can measure M), and a linear standard curve that is routinely used in clinical tests has not been obtained. The method of the present invention has made it possible to measure serum iron at a low concentration as described above with good linearity and high sensitivity with a standard curve having good linearity.

In the present invention, when quantifying serum iron, serum iron bound to transferrin is released to form active aconitase, and isocitrate generated by the reaction of aconitase is oxidized by isocitrate dehydrogenase. Type nicotinamide adenine dinucleotides (hereinafter referred to as NADs) and / or oxidized nicotinamide adenine dinucleotide phosphates (hereinafter referred to as NADs)
Ps) to reduced NADs (hereinafter NADHs) or / and reduced NADPs (hereinafter NADs)
The basic measurement principle is to measure the serum iron amount by detecting the aconitase activity that changes depending on the serum iron concentration as the amount of change to PHs). Specifically, iron bound to transferrin is released in a solution of acidic pH, added to a buffer solution in which at least apo-type aconitase and isocitrate dehydrogenase are dissolved, and apo-type aconitase is converted to holo-type aconitase. However, the amount of serum iron is measured by measuring the production amount or production rate of NADHs and / or NADPHs.

While the Matsubara method, the international standard method, the autoanalyzer method, and the atomic absorption method can obtain only one response to one iron, in the enzymatic assay method of the present invention, the enzyme is amplified. By exhibiting the action, a large number of responses can be obtained for one iron, which leads to high sensitivity and precision. It is also superior to the current measurement method in that it has high specificity for iron. The basic steps of the enzyme assay according to the present invention include the steps of releasing iron from transferrin bound to iron in serum, binding free iron to aconitase, and measuring aconitase activity.
Although it can be decomposed into two basic steps, and iron can be quantified by sequentially performing each step, in particular, in the present invention, each step is combined in such a delicate enzyme reaction. Even though it is sufficiently predicted that the initial purpose will not be achieved, the result of deliberately examining in detail the merging of the respective steps, and after the iron releasing step,
Rather than sequentially performing the step of binding iron to aconitase and the step of measuring aconitase activity independently, by binding both, the aconitase activity was measured while binding the released iron to aconitase, which was totally unexpected. It was confirmed that the enzyme reaction proceeded without any interfering effect and the initial purpose was achieved. The present invention is based on this useful and completely new finding. Therefore, the assay according to the present invention is completed in only two steps, and there are some that have practical value in actual medical fields such as clinical tests. Further, according to it, it is sufficient to prepare two kinds of reagents to be used for measurement, so that errors in the order of use of the reagents are small, and cost reduction can be expected greatly. Hereinafter, the present invention will be described in more detail.

All iron in serum is bound to transferrin. Therefore, in the present invention, iron was released from transferrin in serum and the released iron was measured.

[0010] Therefore, there is provided a method in which iron can be quantitatively released from transferrin in serum, and the release treatment does not adversely affect the subsequent measurement of iron, and the treatment is simple. As a result of diligent research from various fields, we obtained new knowledge that this object can be achieved by setting an acidic atmosphere. Therefore, in the present invention, it is sufficient to bring the releasing step to an acidic pH range,
All methods are available for this purpose, but for example acidic buffers can be used.

Buffers that can be used in the step of releasing serum iron bound to transferrin include acetate buffer, GTA buffer, oxalate buffer, phosphate buffer, glycine buffer, HCl-KCl buffer, potassium diphthalate buffer, Examples include succinate buffer, but the optimum pH of the buffer is 1 to
5, preferably in the range of 2 to 4, and the reagent concentration is 1 to 1,000 mM, preferably 10 to 500.
mM is used and is not particularly specified. If effective, a surfactant, a reducing agent, etc. may be added. Examples of the surfactant include anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, and surfactants having a steroid skeleton.

As the reducing agent usable in the present invention, ascorbic acid, thio-glycolic acid, thio-malate, cysteine, N-acetyl cysteine, hydroxylamine, 2-mercaptoethanol, reduced glutathione,
Dithiothreitol, dithioerythriol, bromide 2-
In addition to aminoethylisothiouronium and thio-glycerol, any substance that promotes binding of iron to aconitase may be used. The reagent concentration is 0-
1,000 mM, preferably 0.1-500 mM is used.

The aconitase which can be used in the present invention is not particularly limited as long as its activity is changed by the addition or non-addition of iron. As a method for adjusting apo-type aconitase, treatment with a chelating agent can be mentioned.
As a chelating agent, ethylenediaminetetraacetic acid (ED
TAs), hydroxyethylethylenediaminetriacetic acid (EDTA-OH), cyclohexanediaminetetraacetic acid (CyDTA), diethylenetriaminepentaacetic acid (DTP)
A), glycol ether diamine tetraacetic acid (GEDT
A), triethylenetetramine hexaacetic acid (TTHA), diaminopropanoltetraacetic acid (DPTA-OH), o-phenanthroline and the like are effective, and those having a background as small as possible in the case where iron is not added are preferable. In addition, ethylenediaminetetraacetic acid (EDTA)
May be a free acid type, a salt type alcohol precipitate such as an alkali, sodium or ammonium salt, or a crystalline substance, and is not particularly limited. The enzyme concentration to add is 0.01
~ 100 u / ml, preferably 0.1-30 u / ml is used.

The isocitrate dehydrogenase which can be used in the present invention is preferably one having a small Km value to isocitrate, but is particularly specified as long as it can react with isocitrate produced by this reaction. There is no such thing. N
It does not matter whether it is specific to ADs or NADPs. The enzyme concentration to be added is 0.01 to 2
00 u / ml, preferably 0.1-20 u / ml is used. Further, isocitrate dehydrogenase and aconitase require magnesium ion or manganese ion in order to express the enzyme activity, but the reagent concentration is 0.5 μM to 100 mM, preferably 5 μM to 10 μM.
Use mM.

The NADs and / or NADPs that can be used in the present invention may be free acid type or salt type alcohol precipitates such as alkali metal or ammonium, or crystalline, and the higher the purity, the more preferable. The known NADs and NADPs can be sufficiently used in the present invention. Not to mention NAD and NADP as NADs and NADPs, thio-NAD and thio-NADP can also be used in the method of the present invention. The method of the present invention can be applied to other NADs and NADPs that can react with isocitrate dehydrogenase. The reagent concentration is 0.01 to 100 mM, preferably 0.1 to 10 mM.

The buffer solution that can be used in the step of binding iron to aconitase and the step of measuring the aconitase activity is not particularly limited, and it may be PIPES buffer, Tris buffer, triethanolamine buffer, glycine buffer, GTA buffer, phosphorus. Acid buffer, borate buffer, etc. may be mentioned, but the optimum pH of the buffer solution may be in the range of 5 to 9, preferably 6 to 8, and the reagent concentration is 1 to 1,000 mM, preferably Use 10-500 mM.

The composition for measuring serum iron of the present invention has a pH of 1 to 1.
5, preferably a first reagent having a pH of 2 to 4 and at least 0.01 to 300 u / ml, preferably 0.1 to 30 u
/ Aconitase and 0.01 to 200 u / ml, preferably 0.1 to 20 u / ml of a second reagent containing isocitrate dehydrogenase, and a reducing agent, NADs or /
And NADPs, magnesium ion or manganese ion is either the first or second reagent, or the first,
It is used by being added over a plurality of second reagents.

As described above, the present invention is a method for quantifying serum iron, which is compatible with highly sensitive fully automated analysis, characterized by quantifying serum iron in a two-reagent system using an enzyme in a reaction system. . The invention also relates to reagents or assay kits for use therein.

[0019]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to measure serum iron with high sensitivity by using an enzyme, can be measured by a two-reagent system, measurement accuracy is improved, and measurement can be performed under mild conditions by using an enzyme. It has a great effect that a rate assay can be performed and the operation is simple. In the present invention, in particular, since it has become possible to perform the measurement with a two-reagent system, the operation is simple and, therefore, operation mistakes are reduced, and therefore, urgent measurement, measurement of a large number of samples, etc. The effect on the ground is huge.

[0020]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0021]

Example 1 Reagent R was prepared according to the composition shown in Table 1 below.
-1, Reagent R-2 was prepared.

[0022]

[Table 1]

[Measurement Method] As the iron standard solution, a 0 to 60 μM ferrous ammonium sulfate solution was used. To 20 μl of the sample (serum sample and iron standard solution), 60 μl of the reagent R-1 was added and stirred, and after incubation at 37 ° C. for 5 minutes,
Reagent R-2 (200 μl) was added, and the mixture was stirred at the same temperature and left standing for 4 minutes, and the amount of change in absorption during 1 minute was measured with a Hitachi 7150 type automatic analyzer. The amount of change in absorbance of a 0 μM ferrous ammonium sulfate solution was subtracted and plotted. The result is shown in FIG. As can be seen in Figure 1, good linearity through the origin was obtained using the method of the invention.

[0024]

Example 2 Reagents R-1 and R-2 used were the same as in Example 1.

[Measurement Method] The measurement method is the same as in Example 1. The number of n is 10. In addition, the coefficient of variation of the serum sample is shown in Table 2 below. As shown in Table 2, the CV value of the serum sample was as good as 3% or less.

[0026]

[Table 2]

[0027]

[Example 3] This measurement method and a kit manufactured by Wako Pure Chemical Industries (Nitro
The serum iron of the sample was measured using both the so-PSAP direct method and Fe-TR). In addition, the reagent R of this measurement method
-1, the reagent R-2 used the same thing as Example 1.

[Measuring Method] The measuring method is the same as in Example 1. The number of samples is 10. The results are shown in Table 3 below. As can be seen from Table 3, the present invention can accurately measure serum iron.

[0029]

[Table 3]

[Brief description of drawings]

1 is a measurement curve of iron measured by the method described in Example 1. FIG.

Claims (17)

[Claims] 1. A method for enzymatically quantifying serum iron, comprising the steps of releasing iron from transferrin in serum and measuring aconitase activity while binding the released iron to aconitase. 2. The method for enzymatically quantifying serum iron according to claim 1, wherein iron is released from transferrin in serum under acidic pH. 3. The method for enzymatically quantifying serum iron according to claim 1, which comprises binding iron released from transferrin to aconitase using a reducing agent. 4. The reducing agent is ascorbic acid, thio-glycolic acid, thio-malate, cysteine, N-acetyl cysteine, hydroxylamine, 2-mercaptoethanol, reduced glutathione, dithiothreitol, dithioerytriol, bromide. The method for enzymatically determining serum iron according to claim 3, which is selected from the group consisting of 2-aminoethylisothiouronium and thio-glycerol. 5. The method for enzymatically quantifying serum iron according to claim 1, wherein the amount of serum iron is measured by detecting aconitase activity with isocitrate dehydrogenase. 6. As a coenzyme for isocitrate dehydrogenase, nicotinamide adenine dinucleotides or /
The method for enzymatically quantifying serum iron according to claim 1, wherein nicotinamide adenine dinucleotide phosphates are used. 7. A reagent for enzymatically measuring serum iron, comprising a step of releasing iron from transferrin in serum and a step of measuring aconitase activity while binding the released iron to aconitase. . 8. The reagent for enzymatically measuring serum iron according to claim 7, which releases iron from transferrin in serum under acidic pH. 9. The reagent for enzymatically measuring serum iron according to claim 8, wherein pH 1 to 5, preferably 2 to 4, is used as the acidic pH. 10. The reagent for enzymatically measuring serum iron according to claim 7, wherein iron released from transferrin is bound to aconitase using a reducing agent. 11. The reducing agent is ascorbic acid, thio-glycolic acid, thio-malate, cysteine, N-acetyl cysteine, hydroxylamine, 2-mercaptoethanol, reduced glutathione, dithiothreitol,
The reagent for enzymatically measuring serum iron according to claim 10, which is selected from the group consisting of dithioerythriol, 2-aminoethylisothiouronium bromide and thio-glycerol. 12. The reagent concentration of the reducing agent is 0 to 1,000.
Reagent for enzymatic determination of serum iron according to claim 11, characterized in that mM, preferably 0.1 to 500 mM is used. 13. The enzyme concentration of aconitase is 0.01
~ 300 u / ml, preferably 0.1 to 30 u / ml is used as the reagent for enzymatically measuring serum iron according to claim 7. 14. The enzyme concentration of isocitrate dehydrogenase is 0.01-200 u / ml, preferably 0.1-2.
The reagent for enzymatically measuring serum iron according to claim 7, wherein 0 u / ml is used. 15. A nicotinamide adenine dinucleotide or / as a coenzyme for isocitrate dehydrogenase
And a reagent for enzymatically measuring serum iron according to claim 14, characterized in that nicotinamide adenine dinucleotide phosphates are used. 16. The coenzyme reagent concentration is from 0.01 to 10.
The reagent for enzymatically measuring serum iron according to claim 15, wherein 0 mM, preferably 0.1 to 10 mM is used. 17. A first reagent of pH 1 to 5, preferably pH 2 to 4, at least 0.01 to 300 u / ml, preferably 0.1 to 30 u / ml of aconitase and 0.0.
1-200u / ml, preferably 0.1-20u / ml
8. The reagent for enzymatically measuring serum iron according to claim 7, which comprises the second reagent containing the isocitrate dehydrogenase.