JP3170066B2 - Unsaturated iron binding ability measurement reagent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent for measuring unsaturated iron binding ability in serum, which can maintain the reagent performance stably for a long time in a solution state.

[0002]

2. Description of the Prior Art The total amount of iron in a living body is about 4000 mg. About 2/3 of them
Is present as hemoglobin iron in red blood cells, and the remaining 1/3
It is stored as ferritin or hemosiderin in tissues such as liver, spleen, and bone marrow. Iron in serum is very small (3-4 mg, about 0.1%) and almost all β ₁
-It binds to transferrin belonging to globulin and flows in blood vessels as serum iron. Transferrin is a carrier of iron in serum mainly produced in the liver, and transports iron and stored iron absorbed from the digestive tract to the bone marrow,
It plays an important physiological role in supplying iron to hemoglobin-producing cells.

[0003] The amount of serum transferrin is clinically indicated by its ability to bind iron. In a normal person, about 1/3 of transferrin binds to iron and is shown as serum iron (SI), and about 2/3 is present in a state not bound to iron and is shown as unsaturated iron binding capacity (UIBC). Have been. Also,
The sum of SI and UIBC, ie, the total amount of transferrin, is indicated by the total iron binding ability (TIBC), and is expressed as “total iron binding ability =
The relationship "serum iron + unsaturated iron binding ability" is established. These measured values of serum iron and unsaturated iron binding ability include various anemias such as iron deficiency anemia, aplastic anemia, pernicious anemia, chronic hemorrhagic anemia, polycythemia vera, infectious anemia, acute hepatitis, chronic anemia, It is of clinical significance in differentiating liver diseases such as hepatitis and cirrhosis.

[0004] As a method for measuring the unsaturated iron binding ability, a known excess amount of iron is added to serum, transferrin and iron are bound, and the remaining iron amount is measured. Is widely used at present because it is suitable for measurement by an automatic analyzer capable of processing many samples accurately and quickly. That is, the binding between transferrin and iron is reversible, strongly dependent on pH, and pH 7-9.
Shows an extremely stable bond, but is easily dissociated by an acidic or strongly alkaline pH 9 or more. Therefore, the measurement of unsaturated iron binding ability is performed by adding an alkaline buffer solution (about pH 7 to 9) containing a certain excess amount of iron ions to serum, thereby binding transferrin and iron, and using a chelating agent for the remaining iron. Colorimetric determination, and subtracting it from the amount of iron added, to calculate it is widely used. In addition, most of the known chelating agents which determine the iron ion concentration in this measurement by colorimetry by chemical coloring are almost specific to ferrous iron, and therefore, when using these, it is necessary to use a reducing agent in combination. Becomes

[0005] As such a reducing agent, there is no bad smell and UI
Ascorbic acid is most widely used in alkaline solutions (pH 7 to 9) necessary for BC measurement because of its strong reducing power. However, since ascorbic acid is unstable in a solution state, it has been difficult to maintain the performance of the UIBC measurement reagent for a long period of time.

[0006] Therefore, a reagent for UIBC measurement that can maintain the reagent performance stably for a long period of time has been desired.

[0007]

Under such circumstances, the present inventors have conducted intensive studies and found that an alkaline buffer containing Fe ²⁺ is used as a first agent, and a chelating agent, ascorbic acid and a stabilizer are contained. Using an acidic solution as the second agent and using them in combination, it has been found that a reagent for UIBC measurement that can maintain the reagent performance stably for a long period of time can be obtained,
The present invention has been completed.

That is, the present invention relates to a first agent having a pH of 7 to 9 containing Fe ²⁺ and a buffer, a chelating agent, ascorbic acid, and a stabilizing agent for ascorbic acid which has a reducing property and is stable in an acidic solution. And a reagent for measuring an unsaturated iron-binding ability, comprising a second agent having a pH of less than 7.

The first agent used in the present invention has a pH of 7 to 9 and contains Fe ²⁺ and a buffer. The Fe ²⁺ source is not particularly limited, and for example, ammonium ferrous sulfate, ferrous chloride, ferrous sulfate, and the like can be used.
It is preferable to mix the first agent in such a manner that the concentration of Fe ^{2+ in} the first agent is 10 to 500 μg / dl, particularly 50 to 200 μg / dl. The buffer is not particularly limited as long as the pH of the first agent can be adjusted to 7 to 9, and for example, Tris buffer, triethanolamine buffer, borate buffer, veronal buffer, phosphate buffer A liquid or the like can be used. The first agent must have a pH of 7 to 9, and within this range, Fe ²⁺ and transferrin can be stably bound.

[0010] The second agent contains a chelating agent, ascorbic acid and a stabilizer and has a pH of less than 7. The chelating agent is not particularly limited as long as it is not easily reduced by ascorbic acid.
(2-pyridyl) -5,6-bis (2- (5-furyl-
Sulfonic acid) -1,2,4-triazine [ferrozine], 3- (2-pyridyl) -5,6-bis (4-phenylsulfonic acid) -1,2,4-triazine [ferren-S], batho Phenanthroline, α, α-dipyridyl and the like. The concentration of these chelating agents in the second agent is preferably 0.5 to 20 mM, particularly preferably 1 to 5 mM. The stabilizer is not particularly limited as long as it has a reducing property and is stable in an acidic solution. For example, dithiothreitol, β-mercaptopropionic acid, N-acetylcysteine, 1-benzyl-1, 4-dihydronicotinamide, sodium bisulfite, α-mercaptopropionic acid, thiourea, (−)-1,4-dithio-L
-Threitol, sodium sulfite, sodium metabisulfite, dl-α-tocopherol acetate and the like. In the second agent, the concentration of the stabilizer is 1 to 100 mM,
Particularly, the concentration is preferably 2 to 50 mM. The concentration of ascorbic acid is 0.02 to 3.0%, particularly 0.05 to 1.0%.
It is preferably 0%.

[0011] The pH of the second agent must be less than 7, preferably 1 to 5, and ascorbic acid can be stably maintained in this range. In the second agent, it is necessary to use ascorbic acid in combination with a stabilizer as a reducing agent, and ascorbic acid alone is liable to be decomposed, and color formation is caused only by the stabilizer. It will be incomplete, making accurate measurement impossible.

In order to measure the unsaturated iron binding ability in serum using the reagent for measuring unsaturated iron binding ability of the present invention, for example, first, a first agent is added to a sample, and transferrin and the first agent in the sample are added.
The Fe ²⁺ in the agent may be bound, and then the second agent may be added thereto to cause the excess Fe ²⁺ to chelate and colorimetrically determine. More specifically, the first agent is added to the serum at 25 to 40 ° C.
After incubation at 25 to 40 ° C. for 2 to 10 minutes, the reaction solution is measured by measuring the absorbance at 570 nm.

[0013]

The reagent for measuring unsaturated iron binding ability of the present invention can stably maintain the reagent performance for a long period of time.

[0014]

EXAMPLES Next, the present invention will be further described with reference to examples, but the present invention is not limited to these examples.

Example 1 A 0.2 M Tris buffer (pH 8.4) containing 5 mg / l ammonium ferrous sulfate was prepared as the first agent, and 0.3%
A 2 mM sodium ferrozine solution (pH 2.3) containing ascorbic acid and 5 mM β-mercaptopropionic acid is prepared as a second agent to obtain a reagent for measuring unsaturated iron binding ability.

COMPARATIVE EXAMPLE 1 A 0.2 M Tris buffer (pH 8.4) containing 5 mg / l ammonium ferrous sulfate and 0.1% ascorbic acid was prepared and used as a first agent to prepare a 2 mM sodium ferrozine solution. To obtain a reagent for measuring unsaturated iron binding ability.

Test Example 1 The unsaturated iron binding ability of the reagents for measuring the unsaturated iron binding ability obtained in Example 1 and Comparative Example 1 was examined. That is, serum 20
After adding 300 μl of the first agent to μl and incubating at 37 ° C. for 5 minutes, adding 75 μl of the second agent and incubating at 37 ° C. for 5 minutes, measuring the absorbance difference at a wavelength of 570 nm, and calculating the unsaturated iron by the following formula The binding ability (UIBC) was determined. The reagent was stored at 10 ° C., and the UIBC value of the same sample was measured over time. Table 1 shows the results. UIBC value = [(Arb−As) / (Ast−Ar)
b)] × 500 (μg / dl), Arb; absorbance when using a physiological saline solution for the sample; Ast; absorbance when using an iron standard solution (500 μg / dl) for the sample; As; Absorbance when using serum)

[0018]

[Table 1]

As is evident from Table 1, when the reagent of Comparative Example 1 was used, the measured value increased after 8 days of storage at 10 ° C., whereas with the reagent of the present invention, even after 400 days. No change in the measured value was observed, and it was confirmed that the performance as a reagent for UIBC measurement was maintained for a long period of time.

Example 2 A 0.2 M Tris buffer (pH 8.4) containing 5 mg / l ammonium ferrous sulfate was prepared as the first agent, and 0.3%
A 3 mM ferren-S solution (pH 2.3) containing ascorbic acid and 5 mM β-mercaptopropionic acid is prepared as a second agent to obtain a reagent for measuring unsaturated iron binding ability.

Test Example 2 For the reagent for measuring the unsaturated iron binding ability obtained in Example 2 and Comparative Example 1, the unsaturated iron bond was examined in the same manner as in Test Example 1.
Table 2 shows the results.

[0022]

[Table 2]

As is clear from Table 2, the reagent of Comparative Example 1 showed an increase in the measured value on the 8th day of storage at 10 ° C., whereas the reagent of the present invention showed an increase even after storage at 10 ° C. for 18 days. No increase in the measured value was observed.

Example 3 A 0.2 M Tris buffer (pH 8.4) containing 5 mg / l ammonium ferrous sulfate was prepared as the first agent, and 0.2%
A 2 mM sodium ferrozine solution (pH 2.3) containing ascorbic acid and 5 mM β-mercaptopropionic acid is prepared as a second agent to obtain a reagent for measuring unsaturated iron binding ability.

Example 4 A 0.2 M Tris buffer (pH 8.4) containing 5 mg / l ammonium ferrous sulfate was prepared as the first agent, and 0.2%
A 2 mM sodium ferrozine solution (pH 2.3) containing ascorbic acid and 5 mM dithiothreitol is prepared as a second agent to obtain a reagent for measuring unsaturated iron binding ability.

Example 5 A 0.2 M Tris buffer (pH 8.4) containing 5 mg / l ammonium ferrous sulfate was prepared as the first agent, and 0.2%
A 2 mM sodium ferrozine solution (pH 2.3) containing ascorbic acid and 5 mM N-acetylcysteine is prepared as a second agent to obtain a reagent for measuring unsaturated iron binding ability.

Test Example 3 The reagents for measuring the unsaturated iron binding ability obtained in Examples 3 to 5 and Comparative Example 1 were stored at 37 ° C., and the UIBC value of the same sample was measured over time in the same manner as in Test Example 1. Was measured. Table 3 shows the results.

[0028]

[Table 3]

As is clear from Table 3, the reagent of Comparative Example 1 showed an increase in UIBC value on the second day of storage at 37 ° C.
While reagent performance cannot be maintained, β-
In the case of the reagent of the present invention using mercaptopropionic acid (Example 3), dithiothreitol (Example 4) or N-acetylcysteine (Example 5), the UIBC value even after 70 days under the extreme conditions of 37 ° C. No increase was observed, confirming that the reagent performance was maintained.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/90 G01N 31/22 G01N 33/50

Claims (2)

(57) [Claims] 1. A first agent having a pH of 7 to 9 containing Fe ²⁺ and a buffer, and a pH 7 containing a chelating agent, ascorbic acid, and a stabilizing agent for ascorbic acid having a reducing property and being stable in an acidic solution. Less than the second agent. 2. The method according to claim 1, wherein the stabilizer is dithiothreitol, β
-Mercaptopropionic acid, N-acetylcysteine,
1-benzyl-1,4-dihydronicotinamide, sodium bisulfite, α-mercaptopropionic acid, thiourea, (−)-1,4-dithio-L-threitol, sodium sulfite, sodium metabisulfite and dl- α-
The reagent for measuring an unsaturated iron binding ability according to claim 1, which is selected from tocopherol acetate.