JPS6232364A - Method for measuring unsaturated iron bonding power in serum - Google Patents

Abstract

PURPOSE:To stabilize ascorbic acid in the co-presence with a color former by stabilizing ascorbic acid as a reducing agent in a soln. state for a long period of time. CONSTITUTION:The ascorbic acid deriv. such as ascorbic acid-2(or 3)-phosphate or ascorbic acid-2(or 3)-sulfonate is made to the reducing agent as the ascorbic acid is liberated after the decomposition by the effect of enzyme such as phosphatase or sulfatase. The long-term stabilization thereof in the co-existence state with the color former is thus made possible.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for quantifying unsaturated iron binding capacity in serum.

Serum iron binds to transferrin, which belongs to β-globulin, and transports iron to erythroblasts in the bone marrow and other tissues that require iron for hemoglobin synthesis.

In normal people, about 1/3 of transferrin binds to iron,
The remaining approximately two-thirds is present in unbound form. Soj de
6) A L et al./F 7-11
Rebellion.

The total amount of iron is called the total iron binding capacity (T I BC), and the amount of iron that can bind to unbound or unsaturated transferrin is called the unsaturated iron binding capacity (U I BC), and this relationship is T
I BC = U I BC ten serum iron.

Trends in serum iron, total and unsaturated iron binding capacity are clinically related to various diseases such as the classification of various anemias, liver diseases, and acute and chronic infections, and their measurement is extremely important clinically.

[Prior Art] Conventionally, the main method for measuring unsaturated iron binding capacity has been to use iron ions and their chelating agents to form a complex, and to quantify the iron ion concentration colorimetrically by chemical coloring. ing. In this method, after adding a known excess amount of iron to serum, the remaining unbound iron is adsorbed and removed using magnesium carbonate and an anion exchange resin, and transferrin-bound iron is measured to determine the total iron-binding capacity. - Determined by subtracting serum iron measured separately, and without using an adsorbent.
The unsaturated iron binding capacity is measured by adding a known amount of iron to the serum to saturate the transferrin in the serum, then colorimetrically quantifying the free iron ion and subtracting it from the added iron ion concentration. be done. Compared to the former method, the latter method is more frequently used because it is suitable for automatic analysis that can accurately and quickly process large amounts of specimens.

Since most of the known coloring agents for colorimetrically measuring iron ion concentration do not form a chelate with trivalent iron, it is necessary to reduce trivalent iron to divalent iron in advance. As reducing agents, thioglycolic acid, ascorbic acid, hydroxylamine hydrochloride, etc. are widely used. In particular, ascorbic acid is most suitable as a reducing agent for iron measurement, but its use has extremely serious problems in terms of stability [JP-A-59-50364].

[Problems to be Solved by the Invention] Conventionally, when ascorbic acid is used as a reducing agent, it is difficult to store it in a solution state for a long period of time, and furthermore, it is difficult to store it in a solution state for a long period of time. It was extremely difficult to stabilize the color former in the coexistence with a color former having a color former. The present invention uses ascorbic acid as a reducing agent,
The purpose of this is to stabilize it in a solution state for a long period of time, and further to stabilize it in the coexistence of a color former having a functional group that is easily reduced.

That is, ascorbic acid is dissolved under acidic conditions (pH 1) in a solution state.
, 5), it is gradually decomposed (oxidized) and has extremely poor storage stability, especially at room temperature (25°C). This means that
This not only makes it difficult for ascorbic acid to coexist with a coloring agent having a functional group that is easily reduced, but also greatly affects the stability of the measurement reagent solution.

In order to solve this problem, a reagent system in which ascorbic acid and a coloring agent are separated may be considered, but in order to simplify the measurement procedure, it is preferable to use a mixture of reagents. . As long as a two-reagent system is desired as a training reagent, the problem of coexistence of trivalent iron and ascorbic acid used as reagents arises. This is because, for example, when trivalent iron coexists with a coloring agent, an increase in the blank is observed, and when it coexists with ascorbic acid, it is reduced to divalent iron, making the reaction with transferrin slow and incomplete. Furthermore, these means cannot provide a substantial solution to the problem of stabilizing ascorbic acid as a reducing agent.

As a result of extensive research into these problems, the present inventors have discovered that these problems can be overcome by using ascorbic acid derivatives, and have completed the present invention.

[Means for Solving the Problems] The present invention uses a 7-scorbic acid derivative in the colorimetric measurement of unsaturated iron binding ability, decomposes this into ascorbic acid by enzymatic action, and converts it into ascorbic acid as a reducing agent for trivalent iron. This is a method for measuring unsaturated iron binding capacity in serum, which is characterized in that it is used as a method for measuring unsaturated iron binding capacity in serum.

Ascorbic acid derivatives in the present invention include ascorbic acid-2 (or 3)-phosphate ester, ascorbic acid-2 (or 3)-containing sulfonic acid ester, ascorbic acid-2 (or 3)-fatty acid monoester, ascorbic acid Examples include acid fatty acid diesters. And the enzymes that act on them include phosphatase, sulfatase, l+11zegorosenin11bar, J/Usokatai7
+ Ascorbic acid produced by salmon 3-batch reduces trivalent iron to divalent one, thereby causing a reaction between divalent iron and the coloring agent. Any known coloring agent may be used, but in particular, 2-nitroso-5-(N-propyl-N-sulfopropylamino)7-ethyl, 2-nitroso-5-(N-ethyl-N-sulfonate) Preferred are propylamine/)7er/9 diFroso7erol derivatives, basso7enanthroline, and the like.

In the measurement method of the present invention, an ascorbic acid N conductor and an enzyme are separately added to a first reagent or a second reagent, and trivalent iron and a coloring agent are separately added to the first reagent or a second reagent. do. Next, a reagent containing trivalent iron is reacted with serum,
After this, a reagent containing a color former is added and reacted. By this method, unsaturated transferrin in serum combines with added iron to become saturated transferrin. At this time,
The unbound iron is reduced to divalent iron by ascorbic acid generated from an ascorbic acid derivative by the action of an enzyme, and is chelated with a coloring agent to produce color. Therefore,
It becomes possible to determine the unsaturated iron binding capacity by subtracting the amount of residual iron that is not bound from the known excess amount of iron that has been added.

[Effect]

The ascorbic acid derivative used in the present invention becomes a reducing agent only after ascorbic acid is liberated after being decomposed by the action of an enzyme, so that it is stable for a long period of time in coexistence with a coloring agent. Furthermore, when ascorbic acid and iron coexist, iron is reduced to divalent and the reaction with transferrin is slow and incomplete, but this is eliminated by the present invention, and iron and reducing agent can coexist. become.

Table 1 shows the stability of the reducing agent of the present invention when conventional ascorbic acid is used as a reducing agent and the stability of the reducing agent of the present invention in an efficacy test. For example, when ascorbic acid is stored at 2 to 8°C, its reducing power decreases by about 50% based on mouth weight after 60 days, whereas the reducing power of the present invention hardly changes. And this comparison is 25
It becomes even more noticeable at ℃.

Table 2 shows the results of a content test comparing the stability of the reducing agent of the present invention with conventional ascorbic acid. Ascorbic acid is 60% at 21 days mouth weight at 2-8℃, 25℃
In contrast, the reducing agent of the present invention does not change even after 60 days under the same conditions.Ascorbic acid was determined by iodometry method (Japanese Pharmacopoeia 10th revision, see ), the time of preparation is set as 100, and the p of the solution is
The pH of l-I was set to around 1.5. On the other hand, the reducing agent in the method of the present invention is used in the liquid chromatography method (column: 4.6φ
×15cm, solution a: 0.08M acetate buffer, 2.8m
It was measured using Mn-hexylamine, 0.1mM EDTA, 2% methanol, detector: UV254n111), and the value at the time of adjustment was set to 100.

Margin below. , −:゛・( □ 1. ,・ Table 1 ~Saichi = ]1 fruit 221° □Ban■ Table 2 苓〉 stomach %〉 Ta %〉 The invention will be explained below by way of examples.

Example 1 Reagent trishydroxylamine 1
9.4g sodium citrate
2. S,? iron alum
8.64■ Swa/-ru AM1
03EX 4.0g alkaline phosphatase 330U/f or more is dissolved in water, adjusted to pH 8.6 with hydrochloric acid, and then adjusted to a total volume of 1,000zf with water.

Second reagent trishydroxylamine 1
9.4g2-nitrone-5-(N-propyl-N-sulfopropylamide/)7enol 1
, 20゜Ascorbic acid sulfate ester magnesium [
Dissolve 0.28g or more (trade name Ninitsucor VC-100, manufactured by Nikko Chemical
6, and then add water to make a total volume of 1,000zz2.

2. Procedure: Add 3xl of the above first reagent to 0.25zl of serum and heat at 37°C.
After incubation for 5 minutes, the second reagent 11
After adding 1 and incubating at 37°C for 20 minutes,
The absorbance of 750r++++ is measured and the unsaturated iron binding capacity (UIBC) value is determined using the following formula.

Calculation formula> ER8: Absorbance when using water as the sample Es: Absorbance when using the specimen (serum) as the sample 1200μg/
dN is the concentration of iron in the first reagent of 3. Results Table 3 shows a comparison of the unsaturated iron binding capacity values determined above with the values determined by the conventional method. As a result, no significant difference was observed between the conventional method and the lvl.

Conventional protein removal methods include Matsubara method for serum iron and Ram5ay method (Medical Technology) for total iron binding capacity.
y t Vol, 11 +No, 10=p, 999
(1983)], the ion exchange resin method was used to measure serum iron using the Fe Neo "Siftest", and the total iron binding capacity was determined by TIBC.
The test was carried out using Neo-Schiftest' (both manufactured by Schiftest Institute), and the value was determined using UIBC=TIBC-serum iron. In addition, the ascorbic acid method was used for measurement based on the same principle as the present invention, and was determined by reducing excess iron (Fe"") with 7scorbic acid657. , Please use the blank space below. Table ■ w PSAP method: 2-nitron-5-(N-propyl-
N-sulfopropylamino)7e/-ruban Method: Baso7enanthroline sulfonate sodium Example 2 1. Reagent 1st Reagent Same as the 1st reagent in Example 1.

Second Reagent The second reagent of Example 1 and 2-nitrone-5-(N-propyl-N-sulfopropylamine 7)phenol were mixed in batho7
Same thing except change to enanthrolin sodium sulfonate.

2. How to operate Same procedure as in Example 1.

3. Results The unsaturated iron binding capacity values determined above are shown in Table 3 in the same manner as in Example 1. As a result, no significant difference was observed between them and the conventional method.

Claims (1)

[Claims] In the colorimetric measurement of unsaturated iron binding ability, an ascorbic acid derivative is used, which is decomposed into ascorbic acid by enzymatic action, and this is used as a reducing agent for trivalent iron.Unsaturated iron in serum How to measure binding capacity.