JPS58144750A - Determination of iron in serum - Google Patents

Abstract

PURPOSE:To achieve an accurate determination of iron in serum by adding a solution of ascorbate oxidase or a solution of oxidizing agent to a serum to measure the absorbancy of a reaction liquid in a reaction treatment of the serum and chromazulol B (CAB). CONSTITUTION:In the treatment of a serum sample with a CAB coloring reagent, a solution of ascorbate oxidase or a solution of oxidizing agent is added to the sample to determine iron in the serum by measuring the absorbancy of the reaction liquid. The oxidizing agent includes patassium folate, sodium folate, etc. The concentration of the oxidizing agent or the ascorbate oxidase added to the reaction liquid is preferably 660mu/l of ascorbate oxidase per 100mg/l of ascorbic acid.

Description

[Detailed description of the invention] This @ Ming relates to a method for quantifying serum iron.

Measurement of iron in serum is clinically important as a test for hemolytic anemia, pernicious anemia, aplastic anemia, acute hepatitis, iron deficiency anemia, and the like. In particular, it is known from recent medical science that transferrin, a biological chelate of iron, increases in iron-deficiency anemia diseases. Diagnostic value: In the living body, iron ingested as a diet is absorbed in the small intestine and is converted to Fe(1) by 7 poferritin in the intestinal wall.
It is oxidized to Karaura and exists in a round shape combined with Ferriti/. When it enters the blood, it is converted into Fe CLKR, further oxidized in the blood, and binds to Fed) and '&) and transferrin. In this way, the absorbed round iron is sent to all parts of the body and maintains homeostasis. Iron in the body is hemoglobin iron, Kenodarobi iron, stored iron,
It exists as serum iron, and the normal concentration of serum iron is 90 to 140 μI/di in adult men and 80 to 120 μI/di in adult women.
l/(11.

Conventional Ojk clear iron measuring methods include colorimetric method, atomic microphotometry, optical emission method (IOP), and electrode method. Among these methods, when applied to automatic biochemical analyzers, the colorimetric method is the simplest and can be analyzed in the same way as other biochemical analysis items, making it ideal for routine testing.

When performing serum iron analysis using a colorimetric method, operations such as those described above are usually required.

(1) Separate iron from transferrin and remove protein.

(2) Fe[) to Fe (1) K-ring yc sul.

f3) Colorize Fe(i) and measure.

In the conventional method, paso7enanthroline, tripyridyltriazine, ferrozine, etc. have been used as the coloring agent in (3), but since all of these react specifically with Fe(1)K, they cannot be used as described above. operations are required,
Previously, due to the low sensitivity of direct measurements, it was difficult to measure with modern automated analyzers that use trace amounts of serum.

We have developed a newly developed highly sensitive iron chelate color former,
Chromazrol B (hereinafter abbreviated as OAB).

0L-1 was obtained by a direct method using
2, serum iron was measured, and good results were obtained (Shimadzu Review Vol. 38, No. 2, pp. 27-3 (1981). The coexistence of ascorbic acid causes errors in the determination of iron, and ascorbic acid is seen in patients who receive infusions and patients who have been administered ascorbic acid complex drugs.Therefore, there are problems with the conventional method. The purpose of the present invention is to provide a method that can accurately and easily quantify iron content even when using blood serum.

The characteristics of the measurement method according to this invention are that serum and OAB
During the reaction treatment, an ascorbic acid oxidase solution or an oxidizing agent solution is added. The negative effects of ascorbic acid are thereby avoided.

Examples of the oxidizing agent used in this invention include potassium iocate and sodium iorate.

Commercially available ascorbic acid oxidase can be used. The concentration of these oxidizing agents in the ascorbic acid oxidase reaction solution varies depending on the concentration of ascorbic acid in the serum.

Generally Ascorbine II 100sF//% I,”
'Fsmar(acid oxidase 666z/#) (preferable. At this added concentration, the assay method of the present invention is sufficient even for normal serum (serum from a patient who is not taking an ascilpiate/acid complex) K. Applicable.

The concentration of the chromazurol B coloring reagent used in this invention may be determined using known conditions.

Further, as described below, in the method of the present invention, it is generally preferred that the ascorbic acid oxidase solution or the oxidizing agent solution be brought into contact with the serum at least simultaneously with or before the chromazurol B coloring reagent. Details are explained in the experimental section.

Experimental part 1, reagent Cromazurol B sodium salt (Dojindo Chemical Laboratory Co., Ltd.). Cetyltrimethylammonium bromide (hereinafter OT)
abbreviated as MA), anhydrous sodium acetate, sodium chloride,
Acetic anhydride, citric acid, sodium citrate, potassium iodate, ascorbic acid oxidase (Biokenka manufactured by Peri/Germa/Heim). (Both use Wako special grade reagents).

2. Reagent adjustment (a) OAB$11; 0AB0.1ftlOOdlxy
Weigh it in a lasco and add distilled water to make it 100m (2.0mm).
ol/l) (b) O'l'MA solution: OTMAO,: 1t100s
Weigh it into a Z/ flask and add distilled water to make it 100s/
8.2 mmol/l).

(0) Acetic acid buffer; anhydrous sodium acetate 251;
Dissolve 110f of sodium chloride in distilled water, and dissolve 8% of acetic anhydride.
．． 0 m? 21 (PH4
,7□5).

(Coloring reagent: Add 90g of OAB solution [60s/KO'l'MA solution, and after mixing, add acetic acid solution to bring the total to 100011t.

(6) Mask reagent: 17f of citric acid (monohydrate) and 35Mt of sodium citrate (dihydrate) - Dissolved in distilled water to make 100IIt.

Coloring reagent for sample blank measurement: Mix coloring reagent and mask reagent at a ratio of 30:1.

(0 potassium iodate solution; potassium iodate 0.31
0.015M-phosphate buffer PH6, 8100g/
The concentration in the reaction reagent was adjusted to 0.311I. The final reaction concentration Fi was set at 0.044-.

(2) Ascorbic acid oxidase solution; ascorbic acid oxidase was diluted with 0.1 M-Li/# sodium buffer pH 5,6, and the concentration in the reaction reagent was 666 u/l.
I adjusted it so that it was K. The final reaction concentration was 133 u/l.

Note that the glassware used for preparing the reagents is soaked overnight in 4-6N hydrochloric acid to remove iron, thoroughly rinsed with distilled water, and carefully dried to avoid contamination. Also, use highly purified ion-exchanged water or distilled water for conditioning.

3. Equipment Shimadzu automatic biochemical analyzer 0L-12 type, Shimadzu atomic absorption/
A flame spectrophotometer AA-640-13 type was used.

4. Analysis Conditions and Procedures Of the 12 channels that can be measured simultaneously under 0L-12, channels 5 and 9 were used for iron analysis and as a sample blank, respectively. The dispenser for reagent delivery is
In order to avoid iron contamination in the flow path, a valve with a Teflon material solenoid valve is used.

(1) A predetermined amount of ascorbic acid was added to the reprinted pooled serum to prepare the sample (see Figure 1 for the amount of ascorbic acid added).
.

50 μm of this serum sample and 200 μm of pure water are dispensed into a reaction tube.

On the other hand, ascorbic acid oxidase solution t-1st reagent was used, and 0.3 d of this was dispensed 15 seconds after dispensing the sample (second row). Furthermore, the coloring reagent (2.d) above is described as the second reagent, and its 1.5 s/l - 0 seconds after dispensing the first reagent (
simultaneous dispensing), 30 seconds (4th column), 60 seconds (6th column)
It was later dispensed.

Historically, in the blank channel, the mask reagent was simultaneously dispensed when the second reagent was dispensed.

(Axis) Instead of the ascorbic acid oxidase solution in the upper ffi, a potassium iodate solution was used as the first reagent and dispensed in the same manner as in (1) above.

(ilil) With IlI light, the maximum absorption wavelength of a colored solution is 630Imn -, but with this -j constant, it is 630Imn -.
Two wavelengths of 28Im and 670Im were used. The reason is,
The detector relationship and absorption curve of 0L-12 are broad, and the absorbance of the reagent plastic is large, so 670 Im
was selected as the target wavelength.

In addition, the sample value correction of measurement data is as follows (1)
This is done automatically based on the formula.

(1) (!=K・(mudomtb)−(St/Sl)
・(A!-mu2b)) Here, 0: Concentration (uf/dl)
, N: 1 degree conversion factor, A1: Analysis channel measurement value,
Mlb "Analysis channel reagent blank, M!: Channel measurement value for sample blank, M, b = Channel reagent blank for sample blank, 8!/81: Sensitivity ratio, (
Al, Alb%A! Calibration was performed using a commercially available control serum Hylant'l (Lot, No. 0368 WOODM). Concentration C is 208 μf/al (Dupont aC
The value obtained as -2,2101 (measured value by &) is -2,2101 (
μf/dJ-mAbs).

5. Analysis Report (1) Figure 1 shows the ascorbic acid removal effect, with the ascorbic acid oxidase-added serum specimen on the horizontal axis and the Fe measurement value on the vertical axis.

For comparison, the results of dispensing pure water are shown by the broken line in Figure 1.
If the second reagent is added at the same time as the first reagent, after 30 seconds, 1
Table 1 shows the effect of O 19 when added after 1 minute, and the simultaneous re-agglomeration data corresponding to these addition conditions are shown in Table 1.

Table 1 Simultaneous reproducibility (1) From the above results, the influence of coexisting ascorbic acid (
It turns out that the negative influence on measured values can be sufficiently eliminated.

Under the above conditions, it is found that it is best to dispense the second reagent 30 seconds after dispensing the first reagent. Table 2 shows the simultaneous reproducibility.

Table 2 Simultaneous reproducibility (2) From the above results, it is clear that the addition of potassium iodate sufficiently avoids the negative effects of coexisting ascorbic acid. i is 3
0 seconds later was IIkIL.

[Brief explanation of the drawing]

FIGS. 1 and 2 are diagrams showing the influence of the addition of ascorbic acid oxidase or potassium sulfate on the measured iron content with respect to the coexistence of ascorbic acid in serum. The vertical axis shows the amount of iron, and the horizontal axis shows the amount of ascorbic acid added.

Claims (1)

[Claims] 1. When treating a serum sample with a Cromazurol B coloring reagent, add an ascorbic acid oxidase solution or a Fi oxidizing agent solution, 1. Measure the absorbance of the reaction solution, and quantify the iron content in the serum. A method for quantifying serum iron consisting of: 2. At the same time as adding ascorbic acid oxidase solution or oxidizing agent solution, add Chromazurol B after about 30 minutes of addition.
The quantitative method according to claim 1, wherein a coloring reagent is added.